Malmö University
Technology and Society
Chemical Engineering
10 credits (C-level) Bachelor Thesis, spring 2005
Identification and determination of NH4+ ions
in “D-Ala-NH2∙HCl” drug substance by
Ion Chromatography (IC)
Zenith Khan and Sara Salim
Instructor: Lena Svensson, PolyPeptide Laboratories, Malmö
Mentor: Monica Lundberg, Malmö Univeristy
Examiner: Johanna Nygren Spanne, Malmö University
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Sammanfattning
Inom kvalitetskontrollavdelningen på PolyPeptide görs bestämningar av negativa joner
(acetat, klorid, jodid etc) med hjälp av jonkromatografi. Sedan en tid har det funnits önskemål
om, att även göra jonkromatografiska bestämningar av positiva joner som ammoniumjonen.
Uppgiften var att ta fram en jonkromatografisk metod för bestämning av ammoniumjoner i
aminosyraderivat, D-Ala-NH2∙HCl som kopplas ihop och bildar peptider. Dessa peptider
används som aktiva läkemedelssubstanser. Det är mest troligt att finna ammoniumklorid i
aminosyraråvaror som innehåller amidgrupp, som D-Ala-NH2∙HCl . Ammoniumklorid bildas
under syntes av dessa aminosyraderivat.
Grunden till att man vill analysera ammoniumjoner är att vid höga halter av
ammoniumklorid i råvaran finns risken att under kemisk peptidsyntes få en oönskad
förorening i produkten som inte är lätt att ta bort. För identifiering av ammoniumjonen
användes en ammoniumstandard.
För att kunna specificera ammoniumtoppen och visa att andra katjoner inte interfererar med
ammoniumjonen användes en katjonstandard som innehöll Li+, Na+, K+, NH4, Mg2+ och Ca2+
joner. Under utveckling av metoden försökte man separera alla dessa toppar med god
resolutionsfaktor, Rs mer än 1.
Utvecklingsfasen har grundat sig på ”trial-and-error” försök. Olika metoder har analyserats
på jonkromatografen och ett underlag för validering har gjorts. Validering innebär att olika
parametrar skall vara innanför vissa gränsvärden. Dessa gränsvärden ska fastställas i ett
protokoll av företaget (PolyPeptide Laboratories) själv.
Med den fastställda metoden som kallats Ad Hoc 345-NH4 cal-6 kan förutom
ammoniumjonen även andra katjoner som Li+, Na+, K+, Mg2+ och Ca2+ joner separeras och
identifieras. Metoden Ad Hoc 345-NH4 cal-6 resulterade i en ammonium standardkurva med
korrelations faktor R2 = 0,999 och visade sig vara lämplig för användning.
De parametrar som bestämts som underlag för validering är detektions- och
kvantifieringsgränserna (LOD och LOQ), specificitets test, linjäritet, noggrannhet,
repeterbarhet och provstabilitet.
Provets (D-Ala-NH2∙HCl) ammoniumhalt bestämdes till 4 %. Alla erhållna resultat för
validerings parametrar har redovisats.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Abstract
The quality control laboratory at PolyPeptide has been performing analysis based on ion
chromatography (IC) of peptides. A method for separation of negative ions (acetate, chloride
etc) is already in use. Lately there has been a need for developing a method to separate
positive ions, like ammonium ions, in peptides.
The task of this project was to develop a method for ion chromatography, suitable for
determination of ammonium ions in the substance D-alanine amide, hydrochloride
(D-Ala-NH2∙HCl). The D-alanine amide, hydrochloride is an amino acid derivative used as
starting material for chemical synthesis of peptides. These peptides are used as active
pharmaceutical substances. It is most possible to find ammonium chloride in amino acids
substances which content amid group, as D-Ala-NH2∙HCl. The ammonium chloride is formed
during the synthesis of these amino acid derivatives.
The reason for analysing ammonium ions is that there is some risk to find undesired
impurities in the product. These impurities are difficult to remove.
A standard solution of six cations which contained the ions (Li+, Na+, K+, NH4+, Mg2+ and
Ca2+ ions) was used to specify the peak of the ammonium ion and to show that the other
cations did not interfere with ammonium ion. These six cations were separated with a good
peak resolution factor, Rs > 1.
The development was based on “trial-and-error” attempts. Different methods have been
analysed by ion chromatography and a foundation of method validation has been determined.
The validation consists of different parameters which shall be within certain limits. These
limits shall be determined in a protocol by the company (PolyPeptide Laboratories) itself.
The best method for separation of ammonium ions was determined to be
Ad Hoc 345-NH4 cal-6. This method is also useful to separate and identify other cations as
Li+, Na+, K+, Mg2+ and Ca2+ ions. The method Ad Hoc 345-NH4 cal-6 resulted in an
ammonium ion standard calibration diagram with a correlation coefficient R2 = 0.999 which
makes this method appropriate for use.
The parameters which have been determined as the foundation of method validation are
limit of detection (LOD), limit of quantification (LOQ), specificity test, linearity, accuracy,
precision and sample solution stability.
The ammonium ion content of the sample (D-Ala-NH2∙HCl) was determined to be 4 %.
The obtained validation parameters are presented in this report.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Acknowledgments
Our work experience at PolyPeptide Laboratories has been very interesting and inspires us
for further work within the pharmaceutical industry in the future. The environment and the
staff have been very co-operative.
We would like to thank our supervisor Lena Svensson for being such a great help with our
work at PolyPeptide Laboratories and for guiding us about the procedures and method used in
the pharmaceutical industry. Lena has put lots of effort and time to help us complete our
thesis.
We would also like to thank our examinator Johanna Nygren Spanne for providing great
amount of help and guidance through out the whole journey of our education at Malmö
University. We very much appreciate Johanna’s kind effort to always be there for us and
encourage us to face difficulties with responsibility.
We are grateful to Monica Lundberg of being helpful with our report and Lars-Åke
Tudesson, for making analytical chemistry seem so interesting and exciting, this goes for all
the teachers at Malmö University for giving us the useful knowledge.
At last, but not the least we thank our families for showing great understanding and always
supporting and encouraging us to complete this work.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Table of contents
Summary……………………………….………...…….. I
Acknowledgments….…….……………………………..II
1.
Introduction ........................................................................................................................ 7
1.1 General ............................................................................................................................. 7
1.2 Aim of this project............................................................................................................ 8
1.3 PolyPeptide Laboratories Group ...................................................................................... 9
1.3.1 Generic peptides ...................................................................................................... 11
2. Background and theory .................................................................................................... 12
2.1 The chemistry of Amino acids and peptides .................................................................. 12
2.2 D-alanine amide, hydrochloride (D-Ala-NH2·HCl) ....................................................... 13
2.2.1 Explanation to why use the amino acid derivative and not the free amino acid in
synthesis of peptides ......................................................................................................... 14
2.2.2 Statement concerning the presence of NH4Cl in starting materials ........................ 14
2.3 The Ion chromatography ................................................................................................ 19
2.3.1 The Columns ........................................................................................................... 21
2.3.1.1 Guard column ............................................................................................... 21
2.3.1.2 Separator column.......................................................................................... 21
2.3.2 The Eluent and the Eluent Generator ...................................................................... 22
2.3.3 The Suppressor ........................................................................................................ 24
2.3.4 The Detector ............................................................................................................ 25
2.3.5 The Software ........................................................................................................... 25
2.4 GMP and validation of analytical procedures ................................................................ 31
2.5 The validation parameters .............................................................................................. 32
2.5.1 Deviations (errors) ................................................................................................... 32
2.5.2 Specificity test ......................................................................................................... 33
2.5.3 Linearity test ............................................................................................................ 33
2.5.4 Precision .................................................................................................................. 33
2.5.4.1 Injection repeatability ................................................................................... 33
2.5.4.2 Analysis repeatability ................................................................................... 33
2.5.4.3 Intermediate precision .................................................................................. 33
2.5.5 Sample solution stability test ................................................................................... 33
2.5.6 Accuracy test ........................................................................................................... 34
2.5.7 Limit of detection (LOD) ........................................................................................ 34
2.5.8 Limit of quantification (LOQ) ................................................................................. 34
3. Method and material............................................................................................................. 35
3.1 Chemicals ....................................................................................................................... 35
3.2 Standard .......................................................................................................................... 35
3.3 Apparatus ....................................................................................................................... 35
3.3.1 Instruments and equipment ..................................................................................... 35
3.3.2 Column and instrument parameters......................................................................... 35
3.4 Procedure ........................................................................................................................ 37
3.4.1 Explanation to the name of the methods ................................................................. 38
3.4.2 Method development ............................................................................................... 38
3.4.3 Preparation of standard solutions for six cations .................................................... 39
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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3.4.4 Preparation of 5 ppm Na+ ion standard solution ..................................................... 40
3.4.5 Preparation of ammonium ion standard solutions ................................................... 41
3.4.6 Preparation of sample (2004-2169) solution in Ad Hoc 345-NH4 cal 3 ................. 42
3.4.7 Preparation of sample (2004-2169) solution in Ad Hoc 345-NH4 cal 6 ................. 43
3.4.8 Isocratic elution by the program Ad Hoc 345 test 3-1 ............................................ 44
3.4.9 Gradient elution by the program Ad Hoc 345 test 2 ............................................... 46
3.4.10 Gradient elution by the program Ad Hoc 345 test 2-3 .......................................... 48
3.4.11 Gradient elution by the two programs Ad Hoc 345 test-lena 1&2 ........................ 49
3.4.12 Gradient elution by the program Ad Hoc 345-NH4 cal-3 ..................................... 51
3.4.13 Gradient elution by the program Ad Hoc 345-NH4 cal-6 ..................................... 53
4. Determination of the validation parameters of Ad Hoc 345-NH4 cal-6 ............................... 55
4.1 Specificity test ................................................................................................................ 55
4.2 Linearity test ................................................................................................................... 56
4.3 Injection repeatability ..................................................................................................... 56
4.4 Analysis repeatability ..................................................................................................... 57
4.5 Intermediate precision .................................................................................................... 57
4.6 Sample solution stability test .......................................................................................... 57
4.7 Accuracy test .................................................................................................................. 58
4.8 Limit of detection (LOD) ............................................................................................... 59
4.9 Limit of quantification (LOQ) ........................................................................................ 60
5. Calculation and formula ....................................................................................................... 61
5.1 Calculated concentration in ammonium ion standard solutions ..................................... 61
5.2 The standard calibration diagram and calculation of ammonium ion content in sample
(2004-2169) solution by Ad Hoc 345-NH4 cal-3 ................................................................. 61
5.3 The standard calibration diagram and calculation of ammonium ion content in sample
(2004-2169) solution by Ad Hoc 345-NH4 cal-6 ................................................................. 63
5.4 The calculation of validation parameters ....................................................................... 65
5.4.1 Calibration diagram for linearity test ...................................................................... 65
5.4.2 Calculation of Injection repeatability ...................................................................... 66
5.4.3 Calculation of Analysis repeatability ...................................................................... 66
5.4.4 Calculation of Intermediate precision ..................................................................... 66
5.4.5 Calculation of Sample stability test ......................................................................... 67
5.4.6 Calculation of Accuracy test ................................................................................... 67
5.4.7 Calculation of LOD ................................................................................................. 69
5.4.8 Calculation of LOQ ................................................................................................. 69
6. Results ................................................................................................................................. 70
6.1 Isocratic elution by the program Ad Hoc 345 test 3-1 ................................................... 70
6.2 Gradient elution by the program Ad Hoc 345 test 2 ...................................................... 71
6.3 Gradient elution by the program Ad Hoc 345 test 2-3 ................................................... 72
6.4 Gradient elution by the program - Ad Hoc 345 test-lena 1&2 ....................................... 73
6.5 Gradient elution by the program Ad Hoc 345-NH4 cal-3 .............................................. 75
6.6 Gradient elution by the program Ad Hoc 345-NH4 cal-6 .............................................. 76
6.7 Result of validation parameters ...................................................................................... 77
7. Discussion and Conclusion .................................................................................................. 79
8. References and Literature ..................................................................................................... 82
9. Abbreviations ....................................................................................................................... 84
Appendix .................................................................................................................................. 85
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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1. Introduction
1.1 General
A major part of financial investments are done in biotech and other pharmaceutical
companies. The biotechnical area is a fast growing and dynamically progressing sector. A lot
has been achieved during the twentieth century, especially the last twenty years. A very good
example is the Human Immunodeficiency Virus (HIV), which is the virus that causes AIDS.
HIV takes over the T-cells and turns them into virus factories that produce thousands of viral
copies. As the virus grows, it damages or kills T-cells, weakening the immune system. This
virus was used to be incurable and caused early death, but with today’s medical treatment this
virus can be controlled and it is practically possible to reduce the death factor. All credit goes
to the fast developing bio technical research.
Manufacturing a product for medical purposes is not just a complicated process but it also
requires huge amount of money, knowledge, research and time. The analytical/analysing part
in developing a pharmaceutical substance is critical to obtain the best and optimal results.
Analysis is performed at every stage of the process. But in pharmaceutical industry the
important analyses are made at a very early stage, where the critical responses can determine
whether the product or substance will function as it was expected or not. These analyses can
be physical, biological or chemical. But in real life some of these analyses depend on each
other and scientists have to work and understand more than just “their” area to solve the
problem. Today controls and analyses (chemical) are made with a very good quality and
accuracy due to the advanced technology and precise instruments.
In the quality control department of PolyPeptide laboratories an ion chromatograph (IC) is
used to determine negative ions as acetate, chloride and iodide. To determine positive ions
like ammonium ion with a cation chromatographic method has been desired for a time. The
focus of this project is on the chemical analysis of the substance
“D-alanine amide, hydrochloride (D-Ala- NH2∙HCl)” which is an amino acid derivative used
as starting material for chemical synthesis of peptides.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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1.2 Aim of this project
The aim of this thesis can be divided in two parts. The first and main part was to develop a
cation chromatographic method for detecting and quantifying ammonium ion in the amino
acid derivative, HCl∙H2N-CH (CH3)-CONH2. D-alanine amide, hydrochloride
The second part includes the determination of basic data for the foundation of method
validation. Parameters such as limit of detection (LOD), limit of quantification (LOQ),
linearity, accuracy, repeatability and the stability of the samples, are to be calculated.
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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1.3 PolyPeptide Laboratories Group
PolyPeptide Laboratories (PPL) Group is one of the world’s leading manufacturers of
custom and generic peptides. The generic peptides are some of the commonly used peptides
for therapeutic applications. A custom made peptide is designed to match the customers
requirement, or an purchase order made by an other company. Being involved in industrial
scale of pharmaceutical peptide manufacturing for over 40 years makes this company a
leading specialist in this particular area. The PolyPeptide Laboratories Group employs more
than 300 people worldwide at manufacturing facilities in Torrance (California), Hillerod
(Denmark), Malmo (Sweden), Prague (Czech Republic) and Wolfenbüttel (Germany). The
facilities in Hillerod and Malmo have good experience in developing solid and solution phase
processes for custom peptides on any scale depending on customers' requirements. The
facility in the USA is devoted mainly to solid phase synthesis.
The Group offers experience in all aspects of solid and solution phase peptide chemistry
and provides a variety of purification techniques, including reverse-phase HPLC and ion
exchange chromatography. Polypeptide Laboratories routinely apply solid phase peptide
synthesis (SPPS), liquid phase peptide synthesis (LPPS) or a combination of both strategies
depending on the nature of the peptide and the quantities of material required. Smaller annual
requirements (gram to kg) are typically manufactured by SPPS for early (pre-Phase III)
clinical development, whereas a LPPS strategy may be needed later for larger batches (1kg100 kg). (22).
Most clinical trials are designated as phase I, II, or III, based on the type of questions that
study is seeking to answer. In Phase III studies, the study drug or treatment is given to large
groups of people (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to
commonly used treatments, and collect information that will allow the drug or treatment to be
used safely
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The quality is very important and that is why the products are manufactured according to
cGMP for Active Substances according to ICH Q7A 1. (21)
Polypeptide Laboratories follows GMP on FDA level, from standard operating procedures
(SOPs) and production documentation to facility design.
Table 1. Regulatory and document for pharmaceuticals purposes and their equivalents in different regions
Country/region Authority
USA
FDA
Regulatory
Document
CFR
§ 210 and
211
Chapter 2
Europe
European Commission
EudraLex
Volume 4
ICH Guidelines
Q7A, Q2A
(GMP)
etc
Pharmaceutical Unit
International
ICH
1
This document (guideline) is intended to provide guidance regarding Good Manufacturing
Practise (GMP) for the manufacturing of Active Pharmaceutical Ingredients (APIs).
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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1.3.1 Generic peptides
Generic peptides have become increasingly important therapeutic agents for the treatment of
a number of conditions, including various types of cancer, hormonal deficiencies and
osteoporosis. PPL offers a comprehensive list of generic peptides, manufactured under cGMP
conditions at industrial scale. The active pharmaceutical ingredients (API) of these peptides
are manufactured in Europe and the US, and sold on the bulk market worldwide.
Here are some of the generic peptides APIs that are produced from PPL.
-
Buserelin is used for the suppression of testosterone in treatment of malignant
neoplasms of the prostate; and as an adjunct to ovulation induction with
gonadotrophins in the treatment of infertility.
-
Calcitonin is the drug of choice for the treatment of osteoporosis, and is also used for
the treatment of hypocalcaemia and Paget’s disease.
-
Vasopressin (Argipressin) is used for the treatment of diabetes. (23)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2. Background and theory
2.1 The chemistry of Amino acids and peptides
The amino acid is a molecule that contains a base and an acid. Molecules like this are
called amfolytes. In general amino acids have the formula of H2N-C(R)H-COOH. Each amino
acid consists of a central tetrahedral carbon atom linked to an amino group, a carboxylic
group and hydrogen. The amino group (NH2-group) of the amino acids functions as the base
and the carboxylic group (COOH-group) as the acid.
All amino acids except for Glycine, are optically active which means that these molecules
are chiral. These are chemically identical but bends polarized light either to the left (L) or
to the right (D). These molecules exist in both forms but the L-form is found to be the more
stable form found in nature. (3)
The mean molecular weight of an amino acid residue is about 110 u (mass unit). A peptide
is formed when two (or more) amino acids are connected. The carboxylic group from one
amino acid reacts with the amino group of another amino acid and forms a peptide bond. The
reaction is a condensation reaction, which means that a water molecule is released/splits off.
See figure 1.
Figure 1: Formation of a peptide bond of two amino acids in a condensation reaction. (26)
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In general the peptides made of small numbers of amino acids are called oligopeptides or
simply peptides. A peptide, which is edified by two amino acids, is called a dipeptide. If more
than 10 amino acids are connected together, that is called a polypeptide. Most naturally
existing polypeptide chains contain between 50 and 2000 amino acid residues and are
commonly referred to as proteins. Proteins are made of amino acids just like peptides. The
difference between the proteins and peptides is in the amount of amino acids. Proteins are
macro molecules with a mass between 6000 and 1 000000 u. (12)
The amino acids can be arranged (placed) in different order and different amounts. There
are 20 different amino acids and depending on their order and structure they form different
proteins. The primary structure refers to the amino acid sequence. The secondary structure
refers to the conformation adopted by local regions of the polypeptide chain. Tertiary
structure describes the overall folding of the polypeptide chain. And the quaternary structure
refers to the association of multiple polypeptide chains to form multi subunit complexes. (3)
2.2 D-alanine amide, hydrochloride (D-Ala-NH2·HCl)
D-alanine amide, hydrochloride is an amino acid derivative used as starting material for
chemical synthesis of peptides. The chemical formula is HCl∙H2N-CH (CH3)-CONH2.
D-alanine amide, hydrochloride is a white powder with a molecule weight of about 124.54 u.
The raw material is durable at 2-8 ºC. (24)
D-Ala-NH2·HCl had a special batch number on PPL “2004-2169”.
According to the sample specification the ammonium ion content in D-Ala-NH2·HCl
(2004-2169) shall be < 3.0 % (w/w). This value was taken from a sample
specification on PPL. (24)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2.2.1 Explanation to why use the amino acid derivative and not the free amino acid in
synthesis of peptides
One almost never uses free amino acids in chemical synthesis; each amino acid has an
amino function and an acid function, as the name states. If two free amino acids where mixed
and a coupling reagent added, then they would link up randomly and in variable chain lengths
- in short polymerise. As this (naturally) is undesirable, one always couple between two
amino acid derivatives, where one has the amino function blocked (called protected, in this
case with a Z-group) and the other has the acid function blocked, e.g. as an amide or an ester.
Amide really is not a protection group, as it is often a desired function in the finished peptide.
So while the coupling to an amide derivative may be the first performed, the amino acid
amide is the last amino acid in the peptide as such.
One does not add the amide function after the coupling to the preceding amino acid. This is
perfectly feasible from a chemical standpoint, but the two ways, in which this can be
performed, have undesirable side-reactions. Treatment of a peptide (ending on an ester) with
ammonia in high concentrations can harm the peptide bonds, and as it never proceeds in a 100
% yield impurities such as a peptide ending on a free acid would have to be removed
afterwards, and this can be tricky and will cost in overall yield. It is much cheaper to have a
purified amino acid amide to start with. (2 & 11)
2.2.2 Statement concerning the presence of NH4Cl in starting materials
In table 2 the chemical formula and the function of each subject “which are concerned in
the chemical reaction during the synthesis of the desire peptide” have been presented
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Table 2. The chemical formula, the abbreviations2 and the function of each subject used in this case are shown.
(13)
Subject
chemical formula
Abbreviation
function
N-hydroxysuccinimide
C4H5NO3
HONSu
coupling
reagent
Di-cyclohexylcarbodiimide
C13H22N2
DCC
coupling
reagent
N-Methylmorpholine
C5H11NO
NMM
organic base
Benzyloxycarbonyl-
(C6H6) -CO-O-NH-R-
Z-3
protection
A benzene ring attached to -CO-O-NH-R-, where
group
NH-R is the amino part of the amino acid
N-Benzyloxycarbonyl-L-
C6H5CH2CH(NHCOOCH2C6H5)COOH
Z-Phe-OH
Z- amino
phenylalanine
N-Benzyloxycarbonyl-L-
acid
C13H15NO4
Z-Pro-OH
Z- amino
proline
Z- phenylalanine amide
acid
C20H23N3O4
Z-Phe-Ala- NH2
intermediate
product
Z- Prolinephenyl amide
C22H25N3O4
L-Alanine methyl ester
hydrochloride
Z-Pro-Phe-NH2
impurity
H-Ala-OMe.HCl
intermediate
product
2
In the IUPAC nomenclatures mentioned above all amino acids have been assigned their own three-letter
abbreviation. Some protection groups often used have also got their own abbreviations, since they are too
cumbersome to write in full all the time. (7)
3
Almost all amino acid are commercially available as Z-derivatives. The sulphur containing ones (Cys and Met)
are not, as the normal removal procedure for Z-groups - catalytic hydrogenation - does not work with these two;
the sulphur inactivates the catalyst. (2)
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NH4Cl in amino acid derivatives
The presence of substantial amounts of NH4Cl or other ammonium salts in amino acid
derivatives used as starting materials for chemical synthesis of peptides is always critical. The
ammonium chloride get deprotonated with a base “which is used during the chemical
coupling reaction of the amino acid derivatives” and forms ammonia. The ammonia will act
as a nucleophil during the chemical coupling resulting in an incorporation of an amide4
function instead of the amino acid required. This side reaction is particularly undesirable
when the peptide has a C-terminal amide function as illustrated below:
Desired reaction:
1. Z-Phe-OH + H-Ala-NH2∙HCl + HONSu/DCC + NMM → Z-Phe-Ala-NH2
+ NMM∙HCl
Side reaction:
2. Z-Phe-OH + NH4Cl + HONSu/DCC + NMM → Z-Phe-NH2 + NMM∙HCl
When proceeding with the coupling step after de-protection (hydrogenation) of the Z-PheAla-NH2 with for example Z-Pro-OH the following two products will be formed:
Z-Pro-Phe-Ala-NH2 (desired product) and Z-Pro-Phe-NH2 (impurity)
The peptide chain is prolonged towards the finished product, which often is at least 10
amino acids long. The longer the desired chain the smaller the difference between impurity
and product will become, and the more difficult it will be to remove it.
The impurity above is called a deletion sequence, which translates into correct sequence
missing one amino acid (can be any position in the chain). These are among the most
undesired impurities, as they are often difficult to detect and remove.
A “normal” deletion sequence at the C-terminal end of a peptide chain can depend on a
coupling reaction not running to completion. In the case above this would result in an
impurity ending on -Pro-Phe-OH, which is not nearly as difficult to remove as an impurity
ending on –Pro-Phe-NH2. The free acid group at the end of the first impurity gives the
impurity very different pH properties compared to the desired peptide, and makes it easier to
4
Amide: R-CO-NH2, an organic compound containing the CONH2 group, closely related to the organic acids
with the COOH grouping. May also be regarded as a derivative of ammonia (NH 3), in which one of the hydrogen
atoms is replaced by an acyl group. (3)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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remove. Therefore deletion sequences of the type, which can be formed with NH4Cl present
in a starting material, are among the most undesired ones.
Unfortunately NH4Cl is most likely to be found in the amide starting materials as
H-Ala-NH2∙HCl. This is due to the way these derivatives are synthesised; generally in the
following way as an example using alanine:
Free alanine (H-Ala-OH) reacts with HCl as catalytic amounts and forms alanine methyl ester
hydrochloride (H-Ala-OMe∙HCl). Afterwards the H-Ala-OMe∙HCl reacts with NH3 in large
excess and forms alanine amide (H-Ala-NH2) and ammonium Chloride (NH4Cl).
1. H-Ala-OH + MeOH + HCl → H-Ala-OMe∙HCl
2. H-Ala-OMe∙HCl + NH3 → H-Ala-NH2 + NH4Cl
Even more NH4Cl is formed during neutralisation of the reaction product. The amino acid
derivative is very hydrophilic and so removing all of the NH4Cl can be a problem for the
suppliers.
Two other facts must also be considered:
I. The molecular weight of NH4Cl is small (53.45 g/mol) compared to that of most
amino acid derivatives and peptides, so even a small content in % (w/w) may represent
a significantly bigger content on a molar basis, and it is this content, which must be
considered in connection with synthesis calculations.
II. Fortunately NH3 is a poorer nucleophil than the α-amino group of an amino acid
derivative, so some help in the form of a better ability to react on the part of the amino
acid derivative is available. Therefore a certain content of NH4Cl can be allowed, but
too much will be a problem.
A reliable method for determining the content of NH4Cl is consequently desirable. Testing
for content of chloride is not a possibility, as the amino acid derivatives ending on an amide
function are almost always available as HCl salts.
Testing for ammonia by conventional pharmacopoeia (USP/Ph. Eur) methods (called limits
tests) showed matrix effects from the amino acid derivative, meaning that the tests were
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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always positive, mainly because they take place at a pH high enough to hydrolyse the amide
function and thus liberating ammonia (NH3).
Ion Chromatography has therefore been the preferred method of analysis, as it is selective and
possesses good sensitivity in the form of a low Limit of Quantification (LOQ). (2 & 11)
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2.3 The Ion chromatography
The Ion Chromatography (IC) apparatus used in this project is made by Dionex. (14)
This instrument uses the latest technology for faster analyze and better resolution of positive
ions. Initially the IC instrument was ordered by PolyPeptide for seperation of negative ions
(anions). In this experiment the same apparatus has been used but some essential parts have
been reordered, such as the coloumns and eluent to convert the IC to separate the cations
instead of anions. Dionex has provided the all the needed hardware and performed a regular
service and installation before attempting the experiments.
According to the Dionex manual ICS-2000 (14) the ion chromatography system typically
consists of a liquid eluent, a high-pressure pump, a sample injector, a guard and separator
column, a chemical suppressor, a conductivity cell and a data collection system, as shown in
figure 2.
The eluent is pumped from the eluent generator through the injection valve to the column.
The exact volume of the sample is also injected through the injection valve. The eluent carries
the sample ions through the separation column “stationary phase” in the ion chromatography
(IC) system. Different samples interact differently and stay for different times in the
stationary phase because different ions have different affinity for the ion exchange pack
material.
Before the ions reach the detector they pass through the suppressor that transforms the
eluent and the ions. The ion chromatography system is equipped with a suppressor that
converts the ions from the solvent into molecules that have no charge, making the signal from
the analyte ion more sufficient. The function of the suppressor is to decrease the background
conductivity of the eluent and to increase the ion sensitivity.
When an ion reaches the detector it increases the voltage between the electrodes in the
detector and a signal will be registered and evaluated on a printer as a chromatogram. (14)
The ion chromatographic separation process can be explained in the following steps. See
Figure 2
1) The eluent is a liquid that helps to separate the sample ions and carries the
sample through the ion chromatography system.
2) The liquid sample is loaded into a sample loop either manually or
automatically. When triggered, the ICS-2000 injects the sample into the eluent
stream. (14)
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2.1) Guard column: The main function of the guard column is to trap
contaminants and remove particles (e.g. carbonate ions in water) that might
damage the separator column. (15)
3) In the separator column the sample ions are separated. The stationary phase
detains the ions until they are separated. (16)
4) The function of the suppressor is to decrease the background
conductivity of the eluent and to increase the ion sensitivity. The suppressor
removes the eluent ions and exchanges the analyte ion for H+ ion (cations), so
that conductivity can be used for detection. (4)
5) By using conductivity detector the ion is detected by concentration
changes, which leads to changes in the conductivity when passing through
the detector. (14)
6) The detector is connected to a printer. When sample ions are passed through
the detector, the conductivity will rise and show a peak in the chromatogram.
(14)
Figure 2: The apparatus and the connected system used in the analysis are shown. (14)
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2.3.1 The Columns
2.3.1.1 Guard column
To protect and increase the lifetime of the separation column, a guard column is often used.
The guard column is a short section of column, is about 0.4 to 1.0 cm long. The guard column
is usually filled with the same stationary phase as the analytical column and attached
immediately in front of it. The guard column acts as a filter and provides some separation as
well. The main function of the guard column is to trap contaminants and remove particles
(e.g. carbonate ions in water) that might damage the separator column. The guard column is
inexpensive and can be changed periodically. (8 & 15)
2.3.1.2 Separator column
In the separator column the sample ions are separated. All separator columns have different
pack material, but they have one thing in common, that they contain charged functional
groups, which are bound to the pack material.
The stationary phase detains the ions until they become separated. Different samples stay
for different time in the stationary phase. The mobility of the ions depends on their mass and
charge. High charge leads to a low dissociation constant and a longer retention time. Large
mass leads to a low mobility and a longer retention time. The condition for separation is that
different ions have different affinity for the ion exchange pack material. The pH of the sample
has an affect on separation. (16)
Ion chromatography is based upon the separation of ions by ion exchange. See figure 3
Figure 3: The ion exchange between the sample cations in the carrier phase and the functional group of the
stationary phase is the essential interaction for separating the cations. This takes place in the separator column.
The equilibrium formula for the ion exchange is:
Resin-COO– H+ + M+
Zenith Khan and Sara Salim
Resin-COO– M+ + H+, (M+ = Cations, ex: NH4+, K+, Na+ ions) (4)
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2.3.2 The Eluent and the Eluent Generator
The eluent is a liquid that helps to separate the sample ions and carries the sample through
the ion chromatography system. The eluent patron used in the separation of cations by IC, is
methane sulfonic acid (MSA) with empiric chemical formula CH3-SO3-H.
The product ion of MSA is methane sulfonate (MSA-) with empiric chemical formula
CH3-SO3-.
The eluent generator generates the desired concentration of the eulent (MSA).
The eluent generator is based on:

The MSA- electrolyte reservoir, low pressure (see figure 4)

The pump

The MSA generator chamber

The Anion exchange connector
In the MSA- electrolyte reservoir there is a Platinum (Pt) cathode where the water
undergoes electrolysis and is reduced to form hydroxide ions and hydrogen gas.
2 H2O + 2e-  2OH- + H2  (g)
Generated OH- displaces MAS- ions and MAS- ions migrate across the anion exchange
connector into the MSA generation chamber.
The pump has been placed before the MSA generation chamber to pump in deionised water
into the MSA chamber.
In the MSA generation chamber a Pt anode is placed. Deionised water is pumped in and
oxidizes at the anode to form hydronium ion (H+) and oxygen gas.
H2O + 2e-  2 H+ + ½ O2  (g)
H+ combined with MAS- ions produce the methanesulfonic acid solution, which is used as the
eluent for cation chromatography.
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A current (DC) is applied between the anode and cathode to control the electrolysis of water
of the device. The applied current is controlled by the given carrier flow rate and the
concentration of MSA. (17)
See figure 4.
Figure 4: The principle of the eluent generator EluGen EGC II MSA. (17)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2.3.3 The Suppressor
The function of the suppressor is to decrease the background conductivity of the eluent and
to increase the ion sensitivity. The suppressor removes the eluent ions and exchanges the
analyte cation for H+ ion, so that conductivity can be used in the detector. (4)
The suppressor used in the IC apparatus is CSRSII, as mentioned in Dionex reference library.
(18)
In the cathode chamber water undergoes electrolysis to form hydrogen gas (H2) and
hydroxide ions (OH-). By electrolysis of water oxygen gas (O2) and hydronium (H+) ions are
formed in the anode chamber. Anion exchange membranes only let negative ions pass through
the membrane. Hydroxide ions move from the cathode chamber into the eluent chamber and
react with hydronium ions and form water.
In the eluent chamber the analyte in dissolved in the eluent (H+ MSA-). Methane sulfonate
(CH3-SO3-) is the anion in the eluent MSA. Methane sulfonate (MSA-) is attracted by an
electrical potential applied to the anode and moves across the membrane towards the anode
and preserves the electric neutrality. The ions (CH3-SO3- and H+) from the system go to the
waste.
After this the analyte is dissolved in water and moves on to the detector. In this way only
sample ions will be detected in the chromatogram. See figure 5 (18)
Figure 5: The chemical reaction in the suppressor. This is auto suppression with cation self-regenerating CSRS
ULTRA II. (18)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2.3.4 The Detector
The conductivity detector is the most common type of detector used in ion
chromatography. This is because the electrolytic conductivity is a universal character among
ions. All ions have the capacity to conduct electron flow (current).
When the sample ions separate they go through the suppressor into a flow cell that is
connected to the detector. Here the ions generate a signal that is detected and evaluated.
The detector is connected to a printer.
The eluate is passed through a flow cell, in which there are two electrodes. Between the
two electrodes there is a voltage. The ability of the solution to transfer electrons increases
when the sample ions reach the cell. The increase of the current is proportional to the increase
of conductivity.
The distance (space) between the electrodes is called d, and the area of the electrodes is
called A.
Quotient d/A is the detector’s cell constant, K [cm-1].
Conductance G between the electrodes is measured continuously and is dependent on the size
of the ion charges and the concentration.
The conductivity, which is an intrinsic character of the solution, can be calculated from the
conductance. In this way, when sample ions pass through the detector, the conductivity will
rise and create a peak in the chromatogram.
Conductance G = 1/R [units: S (Siemens) or Ω-1]
The electrolytic conductivity [S cm-1]
k = K G = (d/ A)*G (25)
2.3.5 The Software
The software system used in this analysis is called Dionex Chromeleon 6.60. (19)
This software program sets all the determining parameters to fixed values for the separation of
the ions. This program contains all the information regarding instrument parameters, for
example the pressure of the pump, the column temperature (see table 5 in 3.3.2) and the
elution concentration during the time. See figure 6.1
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Data Analysis

The conductivity cell transmits the signal to a data collection system.

The data collection system (for the ICS-2000, this is Chromeleon®) identifies the ions
based on retention time, and quantifies each analyte by integrating the peak area or
peak height. The data is quantitated by comparing the sample peaks in a
chromatogram to those produced from a standard solution. The results are displayed as
a chromatogram and the concentrations of ionic analytes can be determined.
Figure 6.1 Shows a typical table of the parameters created by the software, Chromeleon
This creates the program of how the test will be running. After completing the program
description, a sequence is made.
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Sequence is a list where different samples in different order are set for testing. All this is
done through the software Chromeleon. When the samples are put in the sample tray of the IC
apparatus the experiments is ready to run. If several samples are to be processed successively,
they are included in a sample list (sequence table), together with the instrument control and
evaluation information. The sequence collects the data regarding the standard solutions or
sample solutions and how many times of each sample shall be injected.
See the sequence in figure 6.2
Figure 6.2 Shows a sequence created by the software, Chromeleon
The analysis starts as soon as the chromatographic conditions, the samples to process, and
their order have been defined during sequence creation.
Once the program starts to run it generates the chromatograms. The sample preparations,
which are done manually, sequence and the program are the main parts of each testing.
Different tests (experiments) have different set up of sample preparation, program or
sequence.
Sample preparation + Program + Sequence  Method
The method and the development of it will be further explained in 3.4.2 Method
development.
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A report template (chromatogram) contains several special sheets for different print data.
The Peak Analysis and Summary sheets are included in the default report template.
The Peak Analysis tab provides a summary of the characteristics of the single peaks such as
the peak width, peak height, peak type, resolution, asymmetry, and the number of theoretical
plates. Peaks are typically identified by the retention time. In contrast to the peak analysis, the
summary includes all samples in the sequence. The Summary always refers to the current
peak. See the summery table in table 3. (19)
Table 3. Summary of peaks is a part of Chromeleon chromatography software.
Important factors as asymmetry, resolution and plate count of the chromatography analyses, which are defined
below, are calculated:
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Figure 7.1: Chromatogram where tm for the solute peak and tR for the analyte are shown. (5)
The chromatogram in figure 7.1 is described in details below:

The retention time (tR) “time required for the analyte peak to appear” measured in
minutes [min]. tm is the time it would take for an unretained solute peak to appear.(4)

The height of peak measured (by the Chromeleon software) in micro Siemens unit
[µS], (see 2.3.4)

The width of peak measured in cm or mm on the chromatogram. By using the printing
speed (of the printer) the width of the peak is calculated to time [min]

The area bellow the peak measured in micro Siemens  minutes [µS  min]

The resolution (Rs) of two chromatographic peaks is defined by the following formula:
Rs 

1.18( Rt 2  Rt1 )
(W1  W2 )
Rs, the resolution is the retention time measured from time of
injection to time of elution of peak maximum


Rt, the retention time for the analyte peak [min]

W, the width of the peak measured at 50% of the peak height. (4)
The plate count, N is the number of plates of a column toward a particular compound.
Theoretical plate number is a measure of column efficiency. That means how many
peaks can be located per time unit of the chromatogram. (4)
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R 
N  5.54 x  t 
W

2
Asymmetry factor
As = A / B
A + B = W0.1

W0.1 is the Peak widths at 10% of peak height.

A is the widths from tR to the left side.

B is the widths from tR right side of the symmetric peak. (4)
See figure 7.2
Figure 7.2: The Asymmetry of the chromatography peaks is shown. (4)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2.4 GMP and validation of analytical procedures
Good manufacturing practice (GMP) regulations require a quality approach to
manufacturing, enabling companies to minimize or eliminate instances of contamination, mixups, and errors (fitness for use). GMP is regulatory promulgated by the US Food and Drug
Administration (FDA), and contains guidelines to assure high quality of manufacturing active
pharmaceutical ingredients (API). GMP does not ensure a high level safety of using the
product. The international authority, International Conference on Harmonisation (ICH) of
technical requirements of pharmaceuticals for human use, uses guidelines to keep the good
standard of quality. GMP is among those guidelines that are used to support the good quality
work of bio technical processes and pharmaceutical manufacturing. (1 & 20)
The factors that have to be analyzed to validate an analytical process are shown in table 4.
Table 4. Lists typical validation characteristics regarded for the validation of different types of analytical
procedures (6)
Detailed interpretations of the symbols in table 4 are listed below:
-
Signifies that this characteristic is not normally evaluated.
+ Signifies that this characteristic is normally evaluated.
(1) In cases where reproducibility has been performed, intermediate precision is not needed.
(2) Lack of specificity of one analytical procedure could be compensated by other supporting
analytical procedure(s).
(3) May be needed in some cases.
In analytical chemistry there are two very important parameters, which are quality and
reliability. Quality ensure that the analysis fulfil the highly placed requirements and is of
course repeatable.
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(6)
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2.5 The validation parameters
2.5.1 Deviations (errors)
Systematic errors: A systemic error affects the accuracy. This can be for example an
error in the apparatus. (9)
Random errors: A random error affects the repeatability of the analysis. This error is
dependent on the analyst. Practically it could be the dilutions and
weighing of the sample. (9)
Three statistics factors “average, standard deviation and relative standard deviation”
are used in this project.
The average result ( X ): is calculated by summing the individual results and dividing
this sum by the number (N) of individual values. (9)
N
X
X is calculated as in formula 2.
 Xi
1
N
The standard deviation (SD): is a statistic that tells you how tightly all the various
examples are clustered around the mean in a set of data.
With other words the standard deviation is a measure of how
precise the average is, that is, how well the individual numbers
agree with each other. It is a measure of a type of error called
random error. (9)
N
 ( Xi  X )
SD is calculated as in formula 3.
SD =
2
1
N 1
The relative standard deviation (RSD): is often more convenient. It is expressed in
percent and is obtained by multiplying the standard deviation by
100 and dividing this product by the average. (9)
RSD is calculated as in formula 4.
Zenith Khan and Sara Salim
RSD = SD · 100 / mean value
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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2.5.2 Specificity test
The investigation of specificity is performed to show that no peak of other possible
impurities interferes with the ammonium ion peak. (6)
2.5.3 Linearity test
The investigation of linearity is done to show that the analytical procedure, within a given
range, gives test results that are directly proportional to the ion content (in this case NH4+
ion). A correlation coefficient “R2” (how well the experimental point fit a straight line) will be
obtained. (6)
2.5.4 Precision
Precision of the method is a measure of how close the data values are to each other for a
number of measurements under the same analytical conditions. The precision is determined
by injection repeatability, analysis repeatability and intermediate precision. (6)
2.5.4.1 Injection repeatability
Injection repeatability expresses the precision under the same operating conditions over a
short interval of time and with the same solution.
2.5.4.2 Analysis repeatability
Analysis repeatability expresses the precision under the same operating conditions over a
short interval of time
2.5.4.3 Intermediate precision
Intermediate precision expresses within-laboratory variations. It is tested by repeating the
analysis described in the method, by two different analysts. (6)
2.5.5 Sample solution stability test
This investigation is performed to support sample solution stability under normal laboratory
conditions for the duration of the test procedure. This test was done to determine if/how the
ammonium ion content in the sample solution changes over time (6).
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2.5.6 Accuracy test
Accuracy expresses the closeness of agreement between the true value and the value found.
This test was done to observe the closeness of agreement between the true value and the
value found of the ammonium ion content in the sample. (6)
2.5.7 Limit of detection (LOD)
The limit of detection (LOD) is the lowest concentration of analyte in a sample that can be
detected under the stated experimental conditions. The limit of detection is determined as a
signal to noise ratio of 3:1. The noise is determined from a blank injection around the
retention time of NH4+ ion. (6)
2.5.8 Limit of quantification (LOQ)
The limit of quantification (LOQ) is the lowest concentration of analyte in a sample that can
be determined with acceptable precision and accuracy under the stated experimental
conditions. The limit of quantification is determined as a signal to noise ratio of 10:1.The
noise is determined from an injection of a blank solution around the retention time of NH4+
ion. (6)
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3. Method and material
3.1 Chemicals
1 Methanesulfonic Acid (MSA)
2 Milli-Q water
Chemicals of equivalent quality may be used.
3.2 Standard
1 Standard solution of six cations which contained the ions (Li+, Na+, K+,
NH4+, Mg2+ and Ca2+) and the NH4+ ion concentration was almost 400
mg/l or 400 ppm, see the concentration of the other ions in Appendix 1.
2
Ammonium ion standard solution, (concentration: 997 ± 2 µg/ml, see Appendix 7 for
more standard specifications).
3
Na+ ion standard solution, 10 mM NaOH
Standards of equivalent quality may be used.
3.3 Apparatus
3.3.1 Instruments and equipment
1 Autosampler
Dionex AS50
2 Detector and pump
Dionex IC25
3 Eluent generator
Dionex EG40
4 Column oven
Dionex AS50
5 Software system
Dionex Peak Net 6.3 (Chromeleon) or higher
Equivalent equipment may be used.
3.3.2 Column and instrument parameters
The basic parameters such as the following were given fixed values.
All these parameters were the same for the experiments of method development, and therefore
called basic parameters. But in some experiments there had been some changes to optimize
the result 5. See table 5
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Table 5. The fixed value of the IC parameters is shown.
Suppressor
Dionex CSRS-Ultra-II-2mm
Column
Dionex CS12A, 2 mm
Guard column
Dionex CG12A, 2 mm
Trap column
Dionex ATC-1 9 x 24 mm.
4 mm
Lower: 200 bar
Upper: 3000 bar
Pressure limit
Injection volume
25 l
Flow rate
0.25 ml/min.
Suppressor current
50 mA
Temperature5
Auto sampler: 15 °C or 20 °C
Column: 30°C
Area
Peak measurement
5Auto
sampler /tray temperature was not constant in all experiments.
The tray/auto sampler temperature was 15 °C in 3.4.8, 3.4.9, 3.4.10, 3.4.11, 3.4.12.
The tray/auto sampler temperature had only been changed in 3.4.13 (Ad Hoc 345-NH4 cal-6) to 20 °C.
See the cation-exchange column specifications for CS12 in table 6
Table 6. The IonPac CS12A cation-exchange column specifications as mentioned in Dionex reference library.
Dimensions
IonPac CS12A Analytical Column: 2 × 250 mm
IonPac CG12A Guard Column: 2 × 50 mm
Maximum operating pressure
4000 psi (27 MPa)
Mobile phase compatibility
Acidic eluents; 0–100% acetonitrile. Alcohols should
be avoided.
Substrate characteristics
Bead Diameter: 8 µm (2 and 4 mm), 5 µm (3 mm)
Functional group characteristics Ion-Exchange Groups: Grafted carboxylic acid and
phosphonic acid
Capacity
Zenith Khan and Sara Salim
Surface Characteristics: Medium hydrophilic
700 µeq
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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3.4 Procedure
Method development
Gradient elution
Ad Hoc 345 test 2
Ad Hoc 345 test 2-3
Isocratic elution
Using a 0.2 ppm NH4+ ion in
standard solution for six cations
Increasing eluent concentration
(1-35 mM) during 40 minutes
Using a 0.8 ppm NH4+ ion in
standard solution for six cations
Eluent gradient starts and ends at
the same concentration (3 mM)
(3-50 mM) and ending at 3 mM
still during 40 minutes
Ad Hoc 345 test –lena 1& 2
Ad Hoc 345-NH4 cal-3
Ad Hoc 345-NH4 cal-6
Was accepted
Zenith Khan and Sara Salim
Ad Hoc 345 test 3-1
Using a 0.8 ppm NH4+ ion in
standard solution for six cations
Constant eluent concentration
(18 mM) during 40 minutes
Was rejected
Using a 4.0 ppm NH4+ ion in
standard solution for six cations
Eluent gradient starts and ends at
a higher concentration (20 mM)
higher start still during 40
minutes
Using a 0.4 ppm NH4+ ion in standard solution for six cations
Establish a NH4+ ion standard calibration diagram
Determination of NH4+ ion in D-Ala-NH2·HCl
Changing the tray/auto sampler temperature
Establish a new NH4+ ion standard calibration diagram
Determination of NH4+ ion in D-Ala-NH2·HCl
Determination of
validation parameters
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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3.4.1 Explanation to the name of the methods
Regarding to the name of methods like Ad Hoc 345-NH4 cal-6 should be explain that the
Ad Hoc is the term for all investigation works that are analysed outside the GMP at
Polypeptide. Every test is assigned a chronological Ad Hoc number. Our examination work
was the 345:th investigative work at the company and therefore called Ad Hoc 345. The later
designation was an abbreviation that could be easier to save the name of method by the
computer and to remember. For example NH4 represented ammonium ion, cal represented
calibration curve and number 6 represented the number of experiment.
3.4.2 Method development
The results of the program and method were evaluated to either be rejected or accepted.
The method with the acceptable responses was later on validated. The acceptable method is
the method with good separation of different cations and integratable peaks with good
asymmetry factor value (≈ 1) and a tailing factor close to the value of 1.
The optimal eluent (MSA) concentration to carry the cations to the column and separate
them with a high resolution was tested by two different pathways:

isocratic elution: with constant eluent concentration

gradient elution: with increasing eluent concentration
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.3 Preparation of standard solutions for six cations
The standard solution of six cations ((Li+, Na+, NH4+, K+, Mg2+ and Ca2+ ) was bought
from Dionex. This standard solution was used to specify the peak of the ammonium ion and to
show that the other cations did not interfere with ammonium ion. The concentration of NH4+
ions in the standard solution was 400 ppm. To prepare different concentration of NH4+ ions,
the standard solution was diluted with distilled water. See paragraph 3.2 and Appendix 1
for more standard specifications.
The preparation of 0.2 ppm NH4+ ion in standard solution of six cations (std 1)
1.000 ml of the standard solution for six cations was transferred to a 100 ml
flask.
The 100 ml flask was filled to volume with Milli-Q-water and mixed.
1.000 ml of the above solution was transferred to a 20 ml flask.
The 20 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Concentration of NH4+ ion was: 400 ppm / (100 * 20) = 0.200 ppm
The preparation of 0.8 ppm NH4+ ion in standard solution of six cations (std 2)
1.000 ml of the standard solution for six cations was transferred to a 500 ml
flask.
The 500 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Concentration of NH4+ ion was: 400 ppm / 500 = 0.800 ppm
The preparation of 4.0 ppm NH4+ ion in standard solution of six cations (std 3)
1.000 ml of the standard solution for six cations was transferred to a 100 ml
flask.
The 100 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Concentration of NH4+ ion was: 400 ppm / 100 = 4.000 ppm
The preparation of 0.4 ppm NH4+ ion in standard solution of six cations (std 5)
1.000 ml standard solution for six cations was transferred to a 1000 ml flask.
The 1000 ml flask was filled to volume with Milli-Q-water.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
The solution was mixed and filled into a vial.
Concentration of NH4+ ion was: 400 ppm / 1000 = 0.400 ppm
3.4.4 Preparation of 5 ppm Na+ ion standard solution
Stock slution = 10 mM NaOH
MNa / M NaOH = 23.0 /40 = 0.575 %
10 mM NaOH= 0.4 g /1000 mL = 400 mg /l = 400 ppm
400 mg/l · 0.575 = 230 mg/l Na+ ions = 230 ppm Na+ ions
230 ppm ·X assumption ml= 5 ppm ·200 ml
X assumption = 4.35 ml NaOH
4.35 ml NaOH (10 mM) was transferred to a 200 ml flask.
The 200 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.5 Preparation of ammonium ion standard solutions
The ammonium standard was bought from Alltech Associates where the concentration of
ammonium ions was 997 ± 2 µg/ml. The purpose of using this standard was to quantify the
ammonium ions in the sample.
Stock solution
1.00 ml of the NH4+ ion standard solution (997 ± 2 µg/ml) was transferred to a
100 ml flask.
The 100 ml flask was filled to volume with Milli-Q-water.
The solution was mixed.
Standard 0
1.000 ml stock solution was transferred to a 250 ml flask.
The 250 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Standard 1
0.500 ml stock solution was transferred to a 100 ml flask.
The 100 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Standard 2
0.500 ml stock solution was transferred to a 50 ml flask.
The 50 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
Standard 3
2.000 ml stock solution was transferred to a 100 ml flask.
The 100 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and was filled into a vial.
Standard 4
2.000 ml stock solution was transferred to a 50 ml flask.
The 50 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and was filled into a vial.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Standard 5
4.000 ml stock solution was transferred to a 50 ml flask.
The 50 ml flask was filled to volume with Milli-Q-water and mixed.
Standard 6
5.000 ml stock solution was transferred to a 100 ml flask.
The 100 ml flask was filled to volume with Milli-Q-water.
The solution was mixed and filled into a vial.
3.4.6 Preparation of sample (2004-2169) solution in Ad Hoc 345-NH4 cal 3
The sample which was examined was called “2004-2169” by PPL.
See paragraph “2.2” for more information on the sample.
Since the concentration of the ammonium ion was unknown in the sample, the sample was
diluted to different concentrations and tested by different programs. Once the NH4+ ions were
separated and a stable peak was shown in the chromatograms, the sample concentration was
fixed.
This fixed sample concentration was tested by two different programs
“Ad Hoc 345-NH4 cal 3 and Ad Hoc 345-NH4 cal 6” to obtain the optimal separation of
ammonium ions.
20.00 mg (± 0.5 mg) of the sample was weighted into a 20 ml volumetric flask.
The content of this flask was dissolved and filled to volume with Milli-Qwater. The solution was mixed and named “Sample 1a” with a concentration of
1 mg /ml.
1.00 ml of “sample 1a” was transferred to a 100 ml volumetric flask.
The 100 ml flask was filled to volume with Milli-Q-water and mixed (Sample
1b). This sample was used to determine ammonium ions.
The solution (Sample 1b) was filled into a vial.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.7 Preparation of sample (2004-2169) solution in Ad Hoc 345-NH4 cal 6
10.00 mg (± 0.5 mg) of the sample was weighted into a 10 ml volumetric flask.
The content of this flask was dissolved and filled to volume with Milli-Qwater. The solution was mixed (Sample 1a, it had the same name as in 3.4.6
because the concentration was 1 mg/ml).
1.00 ml of Sample 1a was transferred to a 100 ml volumetric flask.
The 100 ml flask was filled to volume with Milli-Q-water and mixed (Sample
1b). This sample was used to determine ammonium ions.
The solution (Sample 1b) was filled into a vial.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.8 Isocratic elution by the program Ad Hoc 345 test 3-1
The development of the method began with isocratic elution trial. During this test the MSA
concentration was constant. The target of this experiment was to determine whether an
isocratic elution should be used to separate the cations or not. The standard solution used in
isocratic elution was std 2 from paragraph 3.4.3, which had a concentration 0.800 ppm
standard solution for six cations.
Figure 7.1: The isocratic elution placed in the flow chart as described in paragraph “3.4 Procedure”
Some of the tests done by Dionex (the makers of the IC used in this project) were also tested
in this project. The column size used at Dionex was 3 mm and in this project the column size
was 2 mm. The isocratic program was called Ad Hoc 345 test 3-1, see Appendix 2.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
The standard solution used in this experiment was standard solution of six cations.
The concentration of ammonium ions in the standard solution of six cations was 400 ppm.
The standard solution was diluted 500 times to get the suitable ammonium ion concentration
for the experiment.
The standard concentration of NH4+ ions corresponds to: 400 ppm / 500 = 0.800 ppm.
This standard concentration was named as “std 2”.
A sequence was prepared with two injections of blanks and four injections of “std 2”. See
Appendix 2
The basic parameters were remained unchanged as in paragraph 3.3.2 Table 5 as listed below.
The tray/auto sampler temperature was 15 °C.
The MSA concentration was 18 mM during the time of 40 minutes. See table 7 and the
program in Appendix 2
Table 7. The change in MSA concentration during time.
MSA (mM)
18
18
18
Time (min.)
0
35
40
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.9 Gradient elution by the program Ad Hoc 345 test 2
The development of the method continued with gradient elution by the program
Ad Hoc 345 test 2, see Appendix 4.
Figure 7.2: The gradient elution placed in the flow chart as described in paragraph “3.4 Procedure”
The target of this experiment was to:

Determine whether the gradient elution should be used to separate the cations or not.

Determine the appropriate concentration of standard solution for six cations by testing
0.2 ppm NH4+ ion in standard solution for six cations.
The standard solution used in this experiment was standard solution for six cations.
See Appendix 1
The concentration of ammonium ions in the standard solution for six cations
was 400 ppm. The standard solution was diluted 2000 times to get the suitable
ammonium ion concentration for the experiment.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
The standard concentration of NH4+ ions corresponds to: 400 ppm / 2000 =
0.200 ppm
This standard concentration was named as “std 1”. See the 3.4.3
A sequence was prepared with two injections of blanks and four injections of “std 1”. See
the sequence in Appendix 4
The basic parameters were remained unchanged as in paragraph 3.3.2 Table 5 as listed below.
The tray/auto sampler temperature was 15 °C.
The MSA concentration was 1-35 mM during the time of 40 minutes. See table 8 and the
program in Appendix 2
Table 8. The change in MSA concentration during time
MSA
1
5
10
15
20
25
30
35
0
05
10
20
25
30
35
40
(mM)
Time
(min.)
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.10 Gradient elution by the program Ad Hoc 345 test 2-3
This program was a further development of gradient elution (see the flow chart in 3.4) of
program Ad Hoc 345 test 2, see Appendix 5.
The target of this experiment was to:

Obtain better separated peaks by changing the gradient.

Stronger concentration of standard solution for six cations to detect all ions (especially
Mg2+ and Ca2+ ions)
The standard solution used in this experiment was standard solution for six cations.
See Appendix 1
The standard concentration of standard solution for six cations used in this experiment was
“std 2”. See 3.4.3
A sequence was prepared with two injections of blanks and eight injections of “std 2”.
See the sequence in Appendix 5
The basic parameters were the same as in 3.3.2. The tray/auto sampler temperature was 15 °C.
The MSA concentration (gradient) was changed more often during the time interval.
The MSA concentration was 3-50 mM during the time of 40 minutes. The gradient ended
with 3 mM.
See table 9 and the program in Appendix 5.
Table 9. The change in MSA concentration during time
MSA
3
5
25
30
35
40
45
45
50
50
3
0
3
10
13
15
20
23
25
30
35
40
(mM)
Time
(min.)
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.11 Gradient elution by the two programs Ad Hoc 345 test-lena 1&2
The program Ad Hoc 345 test- lena 1 & Ad Hoc 345 test-lena 2 (see the flow chart in 3.4)
were almost similar. Both programs began and ended with a MSA concentration about 20
mM. See Appendix 6
The target of this experiment was to:

Detect and determine the peak (cation) in the blank.

Obtain better separated peaks by changing the gradient, with two similar programs.

To detect Mg 2+ and Ca 2+ ions with a stronger concentration of standard solution for
six cations
The standard solution used in this experiment was:

Standard solution for six cations, see 3.2

Na+ ion standard solution, 10 mM NaOH
The concentration of ammonium ions in the standard solution for six cations
was 400 ppm. The standard solution was diluted 100 times to get the suitable
ammonium ion concentration for the experiment.
The standard concentration corresponds to: 400 ppm / 100 = 4.00 ppm
This standard concentration was named as “std 3”, see 3.4.3.
A 5.0 ppm Na+ ion standard solution was prepared as 3.4.4.
In this way the blank (Milli-Q-water) was spiked with 5.0 ppm Na+ ion
standard solution.
See the sequence in Appendix 6
The basic parameters were remained unchanged as in paragraph 3.3.2 Table 5. The tray/auto
sampler temperature was 15 °C.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Ad Hoc 345 test-lena 1: the time duration of 25 minutes. MSA gradient, see table 10
Table 10. The change in MSA concentration during time
MSA (mM)
20
20
40
20
20
Time (min.)
0
15
19
20
25
Ad Hoc 345 test-lena 2: the time duration of 30 minutes. MSA gradient, see table 11
Table 11. The change in MSA concentration during time
MSA (mM) 20
20
40
60
20
20
Time (min.) 0
15
20
25
26
30
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.12 Gradient elution by the program Ad Hoc 345-NH4 cal-3
Program:
Ad Hoc 345- NH4cal-3 (see the flow chart in 3.4 and Appendix 9)
The target of this experiment was to:

Obtain better separated peaks by changing the gradient.

Establish a standard calibration diagram for determining the ammonium ion content of
the sample D-Ala-NH2∙HCl.

Determine the ammonium ion concentration in sample 2004-2169 (D-Ala-NH2∙HCl).
The standard solution used in this experiment was:

Standard solution for six cations, see 3.2

NH4+ ion standard solution, see 3.2
The concentration of ammonium ions in the standard solution for six cations
was 400 ppm.
The standard solution was diluted 1000 times to get the suitable ammonium
ion concentration for the experiment. The standard concentration of NH4+ ions
corresponds to: 400 ppm / 1000 = 0.400 ppm
This standard concentration was named “std 5”. See 3.4.3
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
To establish a standard calibration diagram for determining the ammonium ion content in
the sample D-Ala-NH2∙HCl prepared four different concentrations of NH4+ ion (standard 1, 2,
3 & 4) from NH4+ ion standard solution. See 3.4.5
A standard calibration diagram received when the peak area of all four NH4+ ions standard
solutions were plotted against the different concentration of NH4+ ions. See 5.1 and the
diagram in figure 8 in 5.2.
To determine the ammonium concentration in sample 2004-2169 (D-Ala-NH2∙HCl) a
sample solution as 3.4.6 was prepared.
See the sequence at Appendix 9
The basic parameters were the same as in 3.3.2. The tray/auto sampler temperature was 15 °C.
MSA gradient, see table 12
Table 12. The change in MSA concentration during time
MSA 1
3
5
8
10
12
20
22
23
25
27
28
29
1
3
7
10
12
13
21
23
26
30
35
37
39
43
(mM)
Time 0
(min.)
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
3.4.13 Gradient elution by the program Ad Hoc 345-NH4 cal-6
In this experiment the eluent gradient was analysed by program Ad Hoc 345-NH4 cal-6 (see
the flow chart in 3.4 and Appendix 11).
The target of this experiment was to:

Obtain better separated peaks by changing the gradient.

Establish a standard calibration diagram for determining the ammonium ion content in
the sample 2004-2169 (D-Ala-NH2∙HCl).

Determine the ammonium ion concentration in 2004-2169 (D-Ala-NH2∙HCl).

See if the ammonium ion content of the sample was lower at a higher tray/autosampler temperature.
Figure 7.3: The method Ad Hoc 345-NH4 cal 6 placed in the flow chart as described in paragraph
“4.3 Procedure”.
The standard solution used in this experiment was:

Standard solution for six cations, see 3.2

NH4+ ion standard solution, see 3.2
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
To establish a standard calibration diagram for determining the ammonium ion content in
the sample D-Ala-NH2∙HCl prepared four different concentrations of NH4+ ion (standard1, 2,
3 and 4 in 3.4.5) from NH4+ ion standard solution.
A standard calibration diagram received when the peak area of all four NH4+ ion standard
solutions were plotted against the different concentrations of NH4+ ion. See 5.1 and see the
diagram in figure 9 in 5.3
To determine the ammonium concentration in sample 2004-2169 (D-Ala-NH2∙HCl) a
sample solution (3.4.6) was prepared.
See the sequence at Appendix 11.
The basic parameters were the same as in 3.3.2. The tray/auto-sampler temperature was
changed from 15 ºC to 20 ºC to keep the ammonium ion content in the solutions at a stable
level.
MSA gradient, see table 13
Table 13. The change in MSA concentration during time
MSA 1
3
5
8
10
12
20
22
23
25
27
28
29
1
3
7
10
12
13
21
23
26
30
35
37
39
43
(mM)
Time 0
(min.)
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
4. Determination of the validation parameters of Ad Hoc
345-NH4 cal-6
4.1 Specificity test
The target of this experiment was to calculate the resolution factor (Rs) of cations peaks by
Ad Hoc 345-NH4 cal-6.
A sample (2004-2169) solution spiked with standard solution for six cations was prepared
where the ammonium ion concentration in the spiked sample was 0.5 ppm.
The resolutions between the peaks of six cations were determined.
Preparation of sample (2004-2169) solution
1.00 ml of Sample 1a (in 3.4.7) was transferred to a 50 ml volumetric flask.
The 50 ml flask was filled to volume with Milli-Q-water (Sample 1c).
Sample 1c was spiked with 1 ppm standard solution for six cations.
Preparation of 1 ppm standard solution for six cations
NH4+ ion concentration in the six cation standard was 400 mg/ml or 400 ppm.
The dilution factor: 400 ppm / 1ppm = 400 times
1 ml of the six cation standard (400 ppm) was transferred in a 200 ml flask and
filled to volume with Milli-Q-water. The solution was mixed.
5 ml of the above solution was transferred in a 10 ml flask and filled to volume
with Milli-Q-water and mixed.
Conc standard solution for six cations : 400·5 / 200·10 = 1 ppm
Preparation of the spiked sample
5 ml sample solution was transferred in a 10 ml flask.
The 10 ml flask was filled to volume with 1 ppm standard solution for six
cations.
The solution was mixed and filled into a vial.
The content of the vial was injected twice.
Rs was calculated according to the formula 1 in 2.3.5 by Chromeleon software in summery
of the peaks.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
4.2 Linearity test
The target of this experiment was to obtain a correlation coefficient “R2” value of the
program Ad Hoc 345-NH4 cal-6.
Six solutions were made, whence four of them were the NH4+ ion standard solution
(standard1, 2, 3 and 4 in 3.4.5) used to obtain the calibration diagram in 5.3.
The two other solutions had a concentration of standard 0 and 6 in 3.4.5.
Standard 0 had a lower concentration than standard 1 and standard 6 had a higher
concentration than standard 4.
See the calculated concentration in these six NH4+ ion standard solutions in 5.1
Sequence:
One injection of blank, two injections of each standard solution, see appendix
12
Calculation:
The peak areas of all six NH4+ ion standard solutions were plotted against the
different concentrations of NH4+ ions. See the diagram in figure 10 in 5.4.1
4.3 Injection repeatability
The target of this experiment was to calculate the RSD value when one standard solution
was injected ten times.
Program:
Ad Hoc345-NH4 cal-6, see Appendix 11
An ammonium ion standard solution of concentration 4.985 ·10-5 mg/ml (standard 1) was
prepared according to the method described in 3.4.5. This solution was injected ten times and
the RSD of the peak areas was calculated.
Sequence:
10 injections of ammonium ion standard solution (see injection number 17 to
26 in summary in Appendix 11).
Calculations: The SD and RSD were calculated on 5.4.2.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
4.4 Analysis repeatability
The target of this experiment was to calculate the RSD value when the sample solutions
was injected six times.
Program:
Ad Hoc 345-NH4 cal-6, see Appendix 11
Method:
Six sample solutions were prepared (by Z) according to the method described
in Appendix 10 and the sequence in appendix 11. Each solution was injected
twice and the RSD of the ammonium content in the sample was calculated (see
5.4.3).
Sequence:
See Appendix 11
4.5 Intermediate precision
The target of this experiment was to Calculate the RSD value for n =12, where six of the
samples were prepared by “Z” and six samples by “S”.
Program:
Ad Hoc 345-NH4 cal-6, see Appendix 11
Both analysts (Z and S) prepared six sample solutions each according to the method described
in Appendix 10 and the sequence in Appendix 11. Each solution was injected twice and the
RSD of ammonium ion content in the sample was calculated. See 5.4.4
The peak area used, for each standard, is the mean value of the two injections.
The peak area was used to calculate the ammonium ion concentration, using the linear
equation for the standard calibration diagram, see figure 9 in 5.3.
Sequence:
See Appendix 11
4.6 Sample solution stability test
The target of this experiment was to determine if/how the ammonium ion content in the
sample solution changes in time.
Program:
Ad Hoc 345-NH4 cal-6
Method:
One analyst (Z) prepared six sample solutions according to the method
described in Appendix 10. Each solution was filled in two vials, A and B. The
“B”-vials were kept in 20 ºC for 48 hours before analyzed. The “A” vials were
analyzed right away and vials “B” were analyzed after 48 hrs.
The RSD of ammonium content in the sample was calculated for n=12 (see
5.4.5).
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
The peak area used was the mean value of the two injections.
The peak used to calculate the ammonium concentration, using the linear
equation for the Standard calibration diagram, see figure 9 in 5.3.
All the solutions were injected twice.
Sequence:
See Appendix 11.
4.7 Accuracy test
The target of this experiment was to observe the closeness of agreement between the true
value and the value found of the ammonium ion content in the sample.
Program:
Ad Hoc 345-NH4 cal-6
The idea was to measure the ammonium ion content in the sample. The sample was spiked
with two different ammonium ion standards. One standard should be low in ammonium ion
concentration and one with a high concentration of ammonium ion. In both cases the
ammonium ion content in the samples would obviously increase. The ammonium ion content
in the unspiked sample was subtracted from the spiked sample. The difference should result in
the ammonium ion concentration in the standard solution (the ammonium ion standard used to
spike the sample with) the sample initially was spiked with.
Each solution was injected twice.
The peak area was the mean value of the two injections. The peak area was used to
calculate the ammonium ion concentration, using the linear equation for the Standard
calibration diagram, see figure 9 in 5.3.
The sample solution used in this experiment was the sample solution 1b which
was prepared as in 3.4.6.
Two NH4+ ion standard solutions used in this experiment was
standard 2 and standard 5 which were prepared as in 3.4.4.
Preparation of unspiked sample
5.000 ml of sample solution was transferred in a 10 ml flask.
The 10 ml flask was filled to volume with Milli-Q-water and mixed.
The solution was filled into two vials.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Preparation of the sample spiked with NH4+ ion standard 2
5.000 ml of sample solution was transferred in a 10 ml flask.
The 10 ml flask was filled to volume with NH4+ ion standard 2 and mixed.
The solution was filled into three vials.
Preparation of the sample spiked with NH4+ ion standard 5
5.000 ml of sample solution was transferred in a 10 ml flask.
The 10 ml flask was filled to volume with NH4+ ion standard 5 and mixed.
The solution was filled into three vials.
The content of each vial was injected twice.
Sequence:
See Appendix 11
Calculations: The peak area values were taken from summery in Appendix 11.
See the calculation of accuracy in 5.4.6
4.8 Limit of detection (LOD)
The target of this experiment was to determine LOD. See 2.5.7
Program:
Ad Hoc 345-NH4 cal-6
The blank chromatogram before the lowest NH4+ ion standard concentration (0.04 ppm)
was integrated with the same retention time as for ammonium ion. The noise was determined
from a blank injection around the retention time of NH4+ ion. See the integration data of
injection number 1 (blank) and number 5 (NH4+ ion standard, 0.04 ppm) for the linearity test
in Appendix 12.
Calculated LOD see 5.4.7
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
4.9 Limit of quantification (LOQ)
The target of this experiment was to determine LOQ. See 2.5.8
Program:
Ad Hoc 345-NH4 cal-6
The noise is determined from an injection of a blank solution around the retention time of
NH4+ ion. The blank chromatogram before the lowest NH4 ion standard, 0.04 ppm
concentration was integrated within the same retention time as for ammonium ion. See the
integration data of injection number 1(blank) and number 5 (NH4+ ion standard, 0.04 ppm) for
linearity test in Appendix 12.
Calculated LOQ see 5.4.8
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5. Calculation and formula
5.1 Calculated concentration in ammonium ion standard solutions
C Stock solution = 997 / 100 µg/ml = 0.997 / 100 mg/ml
C standard 0 = 0.997 ·1.0 / (100·250) = 3.988 ·10 -5 mg/ml
C standard 1 = 0.997 ·0.5 / (100·100) = 4.985 ·10 -5 mg/ml
C standard 2 = 0.997 ·0.5 / (100·50) = 9.970 ·10 -5 mg/ml
C standard 3 = 0.997 ·2.0/ (100·100) = 1.994 ·10 -4 mg/ml
C standard 4 = 0.997 ·2.0 / (100·50) = 3.988 ·10 -4 mg/ml
C standard 5 = 0.997 ·4.0 / (100·50) = 7.976 ·10 -4 mg/ml
C standard 6 = 0.997 ·5.0 / (100·100) = 4.985 ·10 -4 mg/ml
5.2 The standard calibration diagram and calculation of ammonium
ion content in sample (2004-2169) solution by Ad Hoc 345-NH4 cal-3
A standard calibration diagram received when the peak areas of four NH4+ ion standard
Solutions “standard 1, 2, 3 and 4” were plotted against the concentration of each NH4+ ion
standard in 5.1. See the peak areas in table 14 or in summery in Appendix 9.
A calibration diagram received when the peak area was a function of ammonium ion
concentration (see the value in table 14). Microsoft Excel software has been used to calculate
the linear regression equation and the correlation coefficient, R2 (see figure 8).
Table 14. The value of ammonium ion standard concentrations and their respective peak areas
Conc NH4+ ion (ppm)
Peak area (µS*min)
0.04985
0.03241
0.09970
0.06806
0.19940
0.17200
0.03988
0.33250
The calibration diagram is shown in figure 8.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
peak area [uS*min]
Standard Calibration Diagram, NH4
y = 0,8707x - 0,0115
R2 = 0,997
0,4
0,3
0,2
0,1
Linjär (Serie1)
0
0
0,2
0,4
0,6
concentration NH4, [ppm]
Figure 8: Standard calibration diagram for NH4+ ion.
This diagram was used to calculate the ammonium ion content in the sample by using its peak
area value.
Peak area of NH4+ ion = 0.33694 [µS·min]
The peak area is the mean value of injection 15 and 16 in summery in
Appendix 9.
The linear regression equation taken from the standard calibration diagram was
used to calculate the concentration from the area of ammonium ion.
The linear regression equation: y = 0.8707x – 0.0115
Where “m” is the intercept and “k” is the slope. In this case
m = -0.0115 [µS·min] and k = 0.8707
x(conc in sample (
mg
y(peak area )  m
)) 
ml
k
The ammonium content could be calculated by using the formula below
where
c = value from calibration curve (mg/mL)
c = 0.40001 mg/L = 4.00·10 -4 mg /mL
msample = sample weight (mg) = 19,95 (mg), see Appendix 8
ContentNH  (%) 
4
c  20  100
 100
msample
Content NH4+ ion (%) = (4.00·10 -4 · 20·100 / (19.95) ·100 = 4.01 %
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5.3 The standard calibration diagram and calculation of ammonium
ion content in sample (2004-2169) solution by Ad Hoc 345-NH4 cal-6
A standard calibration diagram received when the peak areas of four NH4+ ion standard
Solutions “standard 1, 2, 3 and 4” were plotted against the concentration of each NH4+ ion
standard (in 5.1). See the peak areas in table 15 or in summery in Appendix 11.
A calibration diagram received when the peak area was a function of ammonium ion
concentration (see the value in table 15). Microsoft Excel software has been used to calculate
the linear regression equation and the correlation coefficient, R2 (see figure 9).
Table 15. The values of ammonium standard concentrations and their respective peak areas
Conc NH4+ ion (mg/ml) Peak area (µS*min)
4.985 ·10 -5
0.037705
9.970 ·10 -5
0.083835
1.994 ·10 -4
0.177545
3.988 ·10 -4
0.381915
The calibration diagram is shown in figure 9.
Zenith Khan and Sara Salim
Page 63
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Peak area [uS*min]
Calibration diagram, NH4
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
y = 988,79x - 0,0146
R2 = 0,9994
Serie1
Linjär (Serie1)
0
0,0001
0,0002
0,0003
0,0004
0,0005
Concentration [mg/mL]
Figure 9: Standard calibration diagram for NH4+ ion
Peak area of NH4 + ion = 0.406315 [µS·min]
The peak area was the mean value of injection 23 and 24 in summery in
Appendix 11.
The linear equation taken from the standard calibration diagram was used to
calculate the concentration from the area of ammonium ion.
The linear regression equation: y = 988.79x – 0.0146
Where “m” is the intercept and “k” is the slope. In this case
m = -0.0146 [µS·min] and k = 988.79
x(conc in sample (
mg
y (peak area )  m
)) 
mL
k
The ammonium content could be calculated by using the formula below
where
c = value from calibration curve (mg/ml)
c = 4.26·10 -4 mg /mL
msample = sample weight (mg) = 10.04 mg, see Appendix 10 “2004-2169/Z rep
4”
ContetNH 4  ion (%) 
c  10  100
 100
msample
Content NH4+ ion (%) = (4.26·10 -4 ·10·100 / 10.04) ·100 = 4.24 %
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5.4 The calculation of validation parameters
5.4.1 Calibration diagram for linearity test
The peak areas of all six NH4+ ion standard solutions (summery in Appendix 12) were
plotted against the different concentrations of NH4+ion (standard 0, 1, 2, 3, 4&6 in 5.1).
See the peak areas in table 16 or in summery in Appendix 12.
Table 16. The values of six ammonium standard concentrations (0, 1, 2, 3, 4 &6) and their respective peak areas
Conc NH4+ ion (mg/ml) Peak area (µS*min)
3.988 ·10 -5
0.019855
4.985 ·10 -5
0.017435
9.970 ·10 -5
0.077760
1.994 ·10 -4
0.185145
3.988 ·10 -4
0.393345
4.985 ·10 -4
0.511330
The calibration diagram for linearity test is shown in figure 10. The value of R2 was found to
be 0.9993, see figure 10.
.
Linearity of the NH4 standard
Peak area [uS*min]
0,6
y = 1076,5x - 0,0299
R2 = 0,9993
0,5
0,4
Serie1
0,3
Linjär (Serie1)
0,2
0,1
0
0
0,0002
0,0004
0,0006
NH4 conc [mg/ml]
Figure 10: The linearity between the NH4 + ion standard concentrations is shown.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5.4.2 Calculation of Injection repeatability
The peak area values were taken from the 10 injection (number 17 to 26) in summary in
Appendix 11. A mean value of these ten peak areas was used to calculate the ( x ).
N = 10
According to formula 2 in 2.3.5:
X = 0,041418 [µS·min] is mean value of the peak areas
According to formula 3 in 2.3.5:
SD = 2.4895·10-3
According to formula 4 in 2.3.5:
RSD= 6 %
5.4.3 Calculation of Analysis repeatability
The peak area values were taken from the injection (number 17 to 28) in
summary in Appendix 11. The used peak area in this calculation was the mean value of the
two injections. The ammonium ion content in the sample was calculated according to the
same procedure as in 5.3 by Ad Hoc 345-NH4 cal-6.
The X , SD and RSD were calculated according to the formula 1, 2 and 3.
N=6
X = 0.0460 (4.6 %) is the mean value of the ammonium ion content in the
sample.
SD = 2.2635·10-3
RSD= 5 %
5.4.4 Calculation of Intermediate precision
The peak area values were taken from the injection (number 17 to 28 by S and 33 to 44 by
Z) in summary in Appendix 11. The used peak area in this calculation was the mean value of
the two injections. The ammonium ion content in the sample was calculated according to the
same procedure as in 5.3 by Ad Hoc 345-NH4 cal-6.
The X , SD and RSD were calculated according to formula 1, 2 and 3.
N = 12
X = 0.0436 (4.36 %) is the mean value of the ammonium ion content in the
sample.
SD = 3.076 ·10 -3
RSD = 7
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5.4.5 Calculation of Sample stability test
The peak area values were taken from the injection (number 17 to 28 and 63 to 74 by Z) in
summary in Appendix 11. The used peak area in this calculation was the mean value of two
injections. The ammonium ion content in the sample was calculated according to the same
procedure as in 5.3 by Ad Hoc 345-NH4 cal-6.
The X , SD and RSD were calculated according to formula 1, 2 and 3.
N = 12
X = 0.0438 (4.38 %) is the mean value of the ammonium content in the
sample.
SD = 1.989·10-3
RSD = 4.49 %
5.4.6 Calculation of Accuracy test
The peak areas of ammonium ion values were taken from summary in Appendix 11.
Unspiked samples (four injections)
The peak area of ammonium ion was the mean value of injection 46, 47, 48
and 49 in summery in Appendix 11.
Mean value of the area = 0.404585 [µS·min]
Ammonium ion concentration was calculated by using the linear equation from
the standard calibration diagram for NH4+ ion concentration, see figure 9 in
5.3.
C NH4+ ion in
unspiked sample =
4.24 ·10 -4 mg/ml
Samples spiked with ammonium ion standard 2 (six injections)
The peak area of ammonium ion was the mean value of injection 50, 51, 52,
53, 54 and 55 in summery in Appendix 11.
Mean value of the ammonium ion areas = 0.5399 [µS·min]
C NH4+ ion in standard 2 = 9.97·10-5 mg/ml (this was the theoretical value, see 5.1)
The theoretical concentration of ammonium ion in standard 2 in the spiked
sample (theoretical spiked solution) corresponded to:
C NH4+ ion in spiked sample with standard 2 = (9.97·10-5) · 5/10 = 5.00 ·10 -5 mg/ml
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Ammonium ion concentration was calculated by using the linear equation from
the standard calibration diagram for NH4+ ion concentration, see figure 9 in
5.3.
C NH4+ ion in sample spiked with standard 2 = 5.6078 ·10 -4 mg/ml (measured)
Sample + spiked solution:
C NH4+ ion in
unspiked sample +
C NH4+ ion in spiked sample with standard 2:
4.24 ·10 -4 + 5.00 ·10 -5 = 4.74·10 -4 (theoretical)
Sample + spiked solution: 5.6078 ·10 -4 (measured)
% recovery: 5.6078 ·10 -4 / 4.74·10 -4 = 118 %
Samples spiked with standard 5 (mg/mL) (six injections)
The peak area of ammonium ion was the mean value of injection 56, 57, 58,
59, 60 and 61 in summery in Appendix 11.
Mean value of the ammonium ion areas = 0.8076 [µS·min]
C NH4+ ion in standard 5 = 7.976 · 10-4 mg/ml (this was the theoretical value, see 5.1)
The theoretical concentration of ammonium ion in standard 5 in the spiked
sample (theoretical spiked solution) corresponded to:
C NH4+ ion in spiked sample with standard 5 = (7.976 · 10-4) · 5/10 = 3.99·10 -4 mg/ml
Ammonium ion concentration was calculated by using the linear equation from
the standard calibration diagram for NH4 + ion concentration, figure 9 in 5.3.
C NH4+ ion in sample spiked with standard 5 = 8.32 ·10 -4 mg/ml (measured)
Sample + spiked solution:
C NH4+ ion in
unspiked sample +
C NH4+ ion in spiked sample with standard 5:
4.24 ·10 -4 + 3.99•10 -5 = 8.23•10 -4 (theoretical)
Sample + spiked solution: 8.32 •10 -4 (measured)
% recovery: 8.32 ·10 -4 / 8.23·10 -4 = 101 %
Zenith Khan and Sara Salim
Page 68
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
5.4.7 Calculation of LOD
The height of the blank peak was measured (by a ruler) by analyst to be 0.2
cm, where 15 < tr < 18 minutes (read the explanation in 4.8).
The height of the ammonium ion peak (0.04 ppm) was measured (by a ruler)
by analyst to be 5.40 cm where 15 < tr < 18 minutes.
5.40 / 0.20 = 27
This means that the ammonium ion concentration that was used is 27 times
more than the noise.
LOD = 3 · noise
0.04 ppm ↔ 27 times
X ppm ↔ 3 times
X = LOD = 0.04 ·3 / 27 = 0.0044 ≈ 0.004 ppm
The lowest ammonium ion concentration that can be detected should be about
0.004 ppm.
5.4.8 Calculation of LOQ
The height of the blank peak was measured (by a ruler) by analyst to be 0.20
cm, where 15 < tr < 18 minutes (read the explanation in 4.9).
The height of the ammonium peak (0.04 ppm) was measured (by a ruler) by
analyst to be 5.40 cm where 15 < tr < 18 minutes.
5.40 / 0.20 = 27
This means that the ammonium ion concentration that was used is 27 times
more than the noise.
LOQ = 10 · noise
0.04 ppm ↔ 27 times
X ppm ↔ 10 times
X = LOQ = 0.04 ·10 / 27 = 0.015 ppm
The lowest ammonium ion concentration that can be quantified should be
about 0.015 ppm.
.
Zenith Khan and Sara Salim
Page 69
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6. Results
6.1 Isocratic elution by the program Ad Hoc 345 test 3-1
The isocratic elution resulted in very bad peaks. The peaks were not separated and all ions
In the standard solution for six cations were not detectable. One peak (cation) was detected in
the blank with the retention time 5.4 minutes. See the chromatogram for Ad Hoc 345 test 3-1
in figure 11.
14,0
2005-04-26CAT 5 #4 [m odified by XSS]
µS
Std 2
ECD_1
2 - Na - 5,387
12,5
11,3
10,0
8,8
7,5
6,3
5,0
3,8
3 - NH4 - 6,047
2,5
4 - K - 7,447
1 - Li - 4,547
1,3
5 - unkow n5 - 17,040
0,0
-2,0
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
Figure 11: A typical chromatogram of the standard solution for six cations by Ad Hoc 345 test 3-1isocratic.
Different peaks of the cations are shown.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.2 Gradient elution by the program Ad Hoc 345 test 2
The peaks (Li+, Na+, NH4+ and K+ ions) from gradient elution were separated. Mg2+ ion and
Ca2+ ion peaks were not detectable. The retention times for Mg2+ and Ca2+ were between 30
and 35 minutes.
See the chromatogram for Ad Hoc 345 test 2 in figure 12.
One peak (cation) was detected in the blank with the retention time 9.2 minutes.
3,50
2005-04-21cat3 #5 [m odified by Kovalent]
µS
Std 1
ECD_1
2 - Na - 9,213
3,00
2,50
2,00
1,50
1,00
3 - NH4 - 11,493
0,50
4 - K - 14,850
1 - LI - 5,717
-0,00
-0,50
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
Figure 12: A typical chromatogram of the standard solution for six cations by Ad Hoc 345 test 2 gradient.
Different peaks of the cations are shown.
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.3 Gradient elution by the program Ad Hoc 345 test 2-3
The peaks (Li+, Na+, NH4+ and K+ ions) were thinner and bigger due to the high
concentration of standard solution for six cations but Mg2+ and Ca2+ ions peaks were still not
detectable. Stronger standard solution does not affect the retention time of the peaks. One
peak (cation) was detected in the blank with the retention time 10.4 minutes. See the
chromatogram for Ad Hoc 345 test 2-3 in figure 13.
10,0
2005-04-25CAT4 #3 [m odified by LSn]
µS
9,0
Std 2
ECD_1
2 - Na - 10,383
8,0
7,0
6,0
5,0
4,0
3,0
3 - NH4 - 11,643
2,0
4 - K - 13,480
5 - unknow n 4 - 20,787
1 - Li - 8,177
1,0
0,0
-1,0
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
Figure 13: A typical chromatogram of the standard solution for six cations by Ad Hoc 345 test 2-3 gradient.
Different peaks of the cations are shown.
Zenith Khan and Sara Salim
Page 72
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.4 Gradient elution by the program - Ad Hoc 345 test-lena 1&2
The retention time for Na+ ion in the standards solution for six cations and for the peak in
the blank was the same. The peak in the blank was suspected to be Na+ ions. To be sure that
the peak is Na+ ions, a standard of Na+ ions was prepared. The area of the peak in the blank
increased when it was spiked with Na+ ions. The increasing of the area indicated that the peak
detected in the blank was Na+ ion. Ad Hoc 345 test-lena 1 and Ad Hoc 345 test-lena 2 did not
result in better peaks (Li+, Na+, NH4+ and K+ ions). Especially Na+ ion and NH4+ ion peaks
had very low resolution. See the peak analysis in Appendix 6.
Mg 2+ and Ca 2+ ions peaks were better separated in Ad Hoc 345 test-lena 1 and Ad Hoc 345
test-lena 2, compared to previous experiments. Mg 2+ and Ca 2+ ions were detectable, but the
peaks were not completely separated. The Ad Hoc 345 test-lena 2 gradient contained very
high MSA concentration but still the peaks were not separated. See figure 14 and 15.
25,0
2005-04-27CAT6-5PPM #3 [m odified by XSS]
µS
ECD_1
22,5
2 - Na - 5,093
20,0
17,5
15,0
6 - Ca - 13,460
12,5
10,0
3 - NH4 - 5,707
7,5
1 - Li - 4,367
4 - K - 6,953
5,0
2,5
5 - M g - 11,187
0,0
-2,5
-5,0
0,0
m in
5,0
10,0
15,0
20,0
25,0
Figure 14: A typical chromatogram of the standard solution for cations by Ad Hoc 345 test-lena 1 gradient.
Different peaks of the cations are shown.
Zenith Khan and Sara Salim
Page 73
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
25,0
2005-04-27CAT6-5PPM #4 [m odified by XSS]
µS
ECD_1
22,5
2 - Na - 5,087
20,0
17,5
15,0
6 - Ca - 13,417
12,5
10,0
3 - NH4 - 5,700
7,5
1 - Li - 4,360
4 - K - 6,943
5,0
2,5
5 - M g - 11,157
0,0
-2,5
-5,0
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
Figure 15: A typical chromatogram of the standard solution for cations by Ad Hoc 345 test-lena 2 gradient.
Different peaks of the cations are shown.
Zenith Khan and Sara Salim
Page 74
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.5 Gradient elution by the program Ad Hoc 345-NH4 cal-3
All the cation peaks resulted in better separation and asymmetry. See the peak analysis in
Appendix 6. The gradient was accepted for further development. See the chromatogram in
figure 16.
7,00
ECD_1
2005-05-09 PROV-TEST #16 [m odified by XSS]
µS
1 - Na - 14,090
6,00
5,00
4,00
3,00
2,00
1,00
2 - NH4 - 16,270
0,00
-1,00
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
43,0
35,0
Figure 16: Chromatogram of sample 2004-2169
The ammonium ion content in 2004-2169 (D-Ala-NH2∙HCl) was 4 % (more than 3 %).
Zenith Khan and Sara Salim
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.6 Gradient elution by the program Ad Hoc 345-NH4 cal-6
All the cation peaks resulted in better separation and asymmetry. See the chromatogram in
figure 17 and 18
3,50
2005-05-24-VALIDERING-NY-(ANALYS REPETER,REPROD) #13 [m odified by XSS]
µS
ECD_1
4,50
2005-05-24-VALIDERING-NY-(ANALYS REPETER,REPROD) #17 [m odified by XSS]
µS
ECD_1
1 - Na - 13,763
4,00
1 - Na - 13,803
3,00
3,50
2,50
3,00
2,00
2,50
2,00
1,50
1,50
2 - NH4 - 15,577
1,00
1,00
2 - NH4 - 16,040
0,50
0,50
-0,00
-0,50
0,0
-0,00
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
Figure 17: A chromatogram of NH4+ ion
standard solution is shown
43,0
-0,50
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
Figure 18: A chromatogram of sample 2004-2169
is shown.
The standard calibration diagram (figure 9 in 4.3) obtained by running ammonium ion
standard solutions with Ad Hoc 345-NH4 cal-6, had a linear regression of R2 = 0.9994.
The ammonium ion content in 2004-2169 (D-Ala-NH2·HCl) was still found to be more than
3 % (4 %). The temperature change in the auto-sampler did not respond in any specific
difference in ammonium ion content.
Zenith Khan and Sara Salim
Page 76
43,0
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
6.7 Result of validation parameters
Table 17. The result of validation parameters
Specificity test
All the values of peak resolution factor (Rs) were found to be ≥ 1.5,
see the chromatograms number 30 and 31 in Appendix 11.
All peaks of the six cations were separated from each other (with a
good Rs), see figure 19.
Linearity test
The value of R2 was found to be 0.9993, see figure 10
Injection repeatability The calculated RSD value was 6 %
Analysis repeatability The calculated RSD value was 5 %
Intermediate
The calculated RSD value was 7 %
precision
Sample solution
The calculated RSD value was 4.5 %
stability
Accuracy test
% Recovery for sample spiked with std 2 = 118 %
% Recovery for sample spiked with std 6 = 101 %
Limit of detection
approximately 0.004 ppm
(LOD)
Limit of
approximately 0.015 ppm
quantification (LOQ)
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
Chromatogram of specificity test in figure 19
12,0
2005-05-24-VALIDERING-NY-(ANALYS REPETER,REPROD) #31 [m odified by XSS]
µS
ECD_1
2 - Na - 13,800
10,0
8,8
7,5
6,3
5,0
3,8
6 - Ca - 32,630
2,5
3 - NH4 - 15,573
1,3
4 - K - 18,437
5 - M g - 30,233
1 - Li - 11,183
0,0
-2,0
0,0
m in
5,0
10,0
15,0
20,0
25,0
30,0
35,0
43,0
Figure 19: A chromatogram of standard solution for six cations standard is shown.
Zenith Khan and Sara Salim
Page 78
Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
Ion Chromatogrphy (IC)
7. Discussion and Conclusion
The aim of this project was to develop a method to identify and quantify ammonium ion
content in amino acid derivative “D-alanine amide, hydrochloride” as starting material for
chemical synthesis of peptides.
The ground to analyse ammonium ion is that the ammonia group will act as a nucleophil
during chemical coupling reactions resulting in an incorporation of an amide function in stead
of the amino acid required. This side reaction is particularly undesirable. The impurity is
called a deletion sequence, which translates into correct sequence missing one amino acid
(can be any position in the chain). These are among the most undesired impurities, as they are
often difficult to detect and remove.
In the beginning of the method development the instrument parameters like column
temperature, pump pressure and others (see table 5 in 3.3.2) got fixed values according to the
laboratory circumstances by an anion chromatograph used at PPL.
After setting the basic parameters, the next step was to try the experiments to get the optimal
eluent concentration to carry the cations to the column and separate them with a high
resolution.
To get the optimal eluent (MSA) concentration two different pathways were tested:

isocratic elution

gradient elution
Some of the tests done by Dionex (the producers of the IC used in this project) them selves,
were also tested in this project. But due to some apparatus changes e.g. different column size
and different suppressor, the Dionex tests did not work for this project. The column size used
at Dionex was 3 mm and in this project the column size was 2 mm.
The experiment began with separation of cations in a standard solution for six cations.
The peaks (Li+, Na+, NH4+, K+ ions) from gradient elution were better separated compared to
the isocratic elution (see 5.1 and 5.2 ). This gradient elution gave a higher resolution and more
separated peaks than the isocratic elution. The isocratic elution resulted in very bad peaks.
The peaks were not separated and all ions were not detectable (see 5.1).
Therefore the gradient elution was accepted for further experiments separating cations.
One peak (cation) was detected in the blank and was suspected to be Na+ ions. This peak
occurred in every chromatogram where water was used. This suspected peak lead into some
research about the water used at PolyPeptide. The first thought was that this suspected peak
“Na+ ion” was present in the chloride tablets used for purification of water. This problem was
forwarded to Millipore AB, Laboratory Water Division. They responded that the water was
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Identification and determination of NH4+ ions in D-Ala-NH2∙HCl drug substance by
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purified by reversed osmosis membrane. Sodium ions are very small which leads to
difficulties to remove the Na+ ions through the membrane.
To confirm the suspected peak in water, a test was done. In this test water was spiked with
pure Na+ ion solution. This resulted in only one peak that was detected in the chromatogram.
The increase of the peak area confirmed that this must be Na+ ions in the blank.
When a method capable of separating the cations was found, the ammonium ion standard
solutions and the sample were tested. The standards solutions resulted in a calibration diagram
that was used to calculate the ammonium ion concentration in the sample.
According to the sample specification the ammonium ion content in 2004-2169
(D-Ala-NH2∙HCl) shall not be more than 3.0 % (weight/weight). This value was taken from a
sample specification on PPL. The ammonium ion content in 2004-2169 (D-Ala-NH2∙HCl)
calculated by the ammonium ion standard calibration diagram was found to be more than 3 %.
This deviation carried to further research to find out the reason or error of this cause.
The reasons that were found were:

This instability of ammonium ion content in the sample could depend on the variation
of the pH value of the solution. The research showed that the ammonium ion content
in the sample could increase when the sample solution is too acidic or basic. The
optimal pH value must be 3-6. The pH value of the sample solution was found to be
5.03, at room temperature, which is acceptable. The pH value depends on the
temperature of the solution. Lower temperature results in higher pH value. The
chemical reaction that takes place, at a high pH, is when the amide group in the
sample decomposes (hydrolyse) to form ammonia. Fortunately ammonia is a poorer
nucleophil than the α-amino group of an amino acid derivative, so some help in the
form of a better ability to react on the part of the amino acid derivative is available.
Therefore a certain content of NH4Cl can be allowed, but too much will be a problem.

The increase of the ammonium ion concentration in sample could occur due to the
surplus of HCl in the sample. This could change (decompose) the sample into
ammonium chloride and free alanine (more in theory: 2.2).
According to these observations the sample showed to be sensitive to the temperature
changes. That is why the auto-sampler temperature was changed from 15 °C to 20 °C, this
new and the last tested program was called Ad Hoc 345–NH4 cal-6.
By using the method/program Ad Hoc 345–NH4 cal-6 the ammonium ion content in
2004-2169 (D-Ala-NH2∙HCl) was still calculated to be more than 3% (4%).
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The validation parameters are presented in this report. The presented concentration of LOD
and LOQ has not been tested in this report. This method will be validated by PolyPeptide.
But their future planning regarding the validation has not been discussed with the authors of
this report.
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8. References and Literature
Literature
1. Aboul-Enein, Hassan Y. (2001) “Quality and Reliability in Analytical Chemistry”,
chapter 1-8, by CRC Press LLC
2. Bodanszky M.(1993) “ Principles of peptide synthesis”, Springer, 2:th edition
3. Fox Anne Marye & Whitesell James K. ( 1997) “Organic Chemistry”, Sudbury, Mass.
: Jones and Bartlett, 2:th edition, page128 and 820-823
4. Gary D. Christian (2004) “Analytical Chemistry”, Wiley International Edition, 6:th
edition, page 560 – 562 and 622
5. Gary D. Christian (2004) “Analytical Chemistry”, Wiley International Edition, 6:th
edition, Figure 19.3, page 561
6. ICH Steering Committee (27 October 1994) “Text on Validation of analytical
Procedures, Q2A”, ICH Harmonised Tripartite Guideline
7. IUPAC-IUB Joint Commission on Nomenclature. (1984) “Biochem. J.”, page 219,
345-373
8. Meloan Clifton E. ( 1999) “Chemical separations”, Wiley & Sons INC, chapter 19,
page 198-199 ( gard
9. Miller, JN, & Miller, JC. (2000) “Statistics and Chemometrics for Analytical
Chemistry”, 4:th edition, page 3-7, 21& 22
10. Small Hamish (1 Nov 1989) “Ion Chromatography”, Springer
11. Statement Sørensen A.H. (20 September 2005) “Statement concerning the presence of
NH4Cl in starting materials”, PolyPeptide Laboratories
12. Stryer L. (2002) “Biochemistry”, W.H Freeman And Company, 5:th edition
Internet Website
13. ChemExper Chemical “Find chemicals in the ChemExper Chemical
Directory”, 2006-04-10
http://www.chemexper.com/index.shtml?main=http://www.chemexper.com/search/cas
/538-75-0.html
14. Dionex Corporation “ICS-2000 Ion Chromatography System operator’s manual”,
March 2003, Document No. 031857-01, page 13-16
http://www1.dionex.com/en-us/webdocs/4568_31857-01_ICS2000_Operators_V19.pdf
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15. Dionex Corporation (September 2004)“CG12A Guard column in IC ”,Dionex
reference Library (CD-rom), product catalogue, http://www.kovalent.se
16. Dionex Corporation (September 2004)“CS12A Separator column in IC ”,Dionex
reference Library (CD-rom), product catalogue, http://www.kovalent.se
17. Dionex Corporation (September 2004) “EG50 Eluent Generator System Manual”,
Dionex reference Library (CD-rom), Document No. 031908-02, Figure 2, page 7 of
66, http://www.kovalent.se
18. Dionex Corporation (September 2004) “Suppressor SRS ULTRA II Manual”, Dionex
reference Library (CD-rom), Document No. 031956-03, Figure 3B, page 7 of 50,
http://www.kovalent.se
19. Dionex Corporation “Chromeleon Chromatography Management System- Tutorial
and user manual”, November 2002,
http://www1.dionex.com/en-us/webdocs/4569_CM650_V18.pdf
20. Federal Food, Drug, and Cosmetic Act “What Is GMP?”, 2005-03-04
http://www.gmp1st.com/gmp.htm
21. PolyPetide Laboratories “GMP Compliance & Regulatory Support”, 2005-03-05
http://www.polypeptide.com/regado.jsp?type=page&id=98
22. PolypePtide Laboratories “PolyPeptide Laboratories Group”, 2005-03-05
http://www.polypeptide.com/regado.jsp?type=page&id=85
23. PolypePtide Laboratories “Generic peptides”, 2005-03-05
http://www.polypeptide.com/regado.jsp?type=page&id=114
24. Senn Chemicals AG, “sample specification for D-Ala-NH2∙HCl”, January 2002
http://www.sennchem.com
25. SeQuant AB, Inovators in hemical AnalysisTM “Jonkromatografi I praktiken”,
2005-04-01
http://www.sequant.com/products/cars/pdf/SeQuant_Jonkromatografi_i_Praktiken.pdf
26. Svenska biotermgruppen “Peptide bond”, 2005-04-01
http://www.tnc.se/bioterm/bilder/dp.gif
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9. Abbreviations
API: Active Pharmaceutical Ingredients
CFR: Code of Federal Regulations (USA)
cGMP: Current Good Manufacturing Practice
EudraLex: European laws and regulations regarding Pharmaceutical
FDA: U.S. Food and Drug Administration
GMP: Good Manufacturing Practice
HPLC: High performance Liquid Chromatography
ICH: International Conference on Harmonisation of technical requirements for registration of
pharmaceuticals for human use.
IEC: Ion Exchange Chromatography
KI: Confidence Interval
LOD: Limit of Detection
LOQ: Limit of Quantification
LPPS: Liquid Phase Peptide Synthesis
MSA: Methane Sulfonic Acid
MSA-: Methane Sulfonate
PPL: PolyPeptide Laboratories
PPM: Parts Per Million
Q7A: Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients
Q2A: Text on Validation of Analytical Procedures
SOP: Standard Operating Procedure
R2: Linear Regression
RSD: Relative Standard Deviation
S: Sara
SD: Standard Deviation
SPPS: Solid Phase Peptide Synthesis
Std: Standard solution
Z: Zenith
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Appendix
Appendix 1: Six Cation Standard Certificate----------------------------------------------------------1
Appendix 2: Ad Hoc 345 test 3-1-----------------------------------------------------------------------2
Appendix 3: Isocratic Elution by Dionex-------------------------------------------------------------- 5
Appendix 4: Ad Hoc 345 test 2------------------------------------------------------------------------- 6
Appendix 5: Ad Hoc 345 test 2-3----------------------------------------------------------------------10
Appendix 6: Ad Hoc 345 test-lena 1, Ad Hoc 345 test-lena 2-------------------------------------14
Appendix 7: Ammonium ion Standard Certificate-------------------------------------------------- 20
Appendix 8: Sample 2004-2169 using in Ad Hoc 345-NH4 cal-3-------------------------------- 22
Appendix 9 Ad Hoc 345-NH4 cal-3------------------------------------------------------------------- 23
Appendix 10: Sample 2004-2169 using in Ad Hoc 345-NH4 cal-6-------------------------------28
Appendix 11: Ad Hoc 345-NH4 cal-6-----------------------------------------------------------------30
Appendix 12: Linearity test (validation)--------------------------------------------------------------43
Appendix 13: Chemicals safety precautions--------------------------------------------------------- 47
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