Appendix: Parecoxib and valdecoxib measurement by ultra performance liquid
chromatography-tandem mass spectrometry (UPLC-MS/MS)
Samples of parecoxib sodium and valdecoxib used as standard materials in the assay
were a gift from Pfizer Australia Pty Ltd, North Ryde, Australia.
Parecoxib and
valdecoxib were extracted from 50 μL of milk by protein precipitation with 1 mL of
methanol containing valdecoxib-13C215N (2 g/L) as internal standard (Toronto
Research Chemicals Inc, North York, ON, Canada).
A 20 μL aliquot of the
supernatant was injected onto the UPLC-MS/MS. For extraction of parecoxib and
valdecoxib from plasma, it was necessary to use a liquid-liquid extraction procedure.
Aliquots of plasma (250 μL) and internal standard (50 μL of valdecoxib-13C215N; 100
ng/mL) were extracted with 100 μL of 1M HCl and 5 mL of dichloromethane.
Because parecoxib is an acidic drug, extraction at low pH results in adequate
recovery. Although valdecoxib is basic, the formation of a chloride salt, soluble in
dichloromethane accounts for adequate recovery of both valdecoxib and the internal
standard in the above conditions. After centrifugation (2,000 g for 5min) the top
aqueous layer was aspirated to waste. The dichloromethane layer was transferred to
clean glass tubes, and dried with a gentle stream of N2 at 50oC. The residue was
reconstituted with 1 mL of methanol and 20 μL aliquots were injected onto the UPLCMS/MS system.
The UPLC-MS/MS system consisted of a Waters Acquity UPLC (Milford, MA, USA)
coupled with a Waters Premier XE MS-MS (Milford, MA, USA).
The LC mobile
phases were (A) 25% acetonitrile in 2 mM ammonium acetate / 0.1% formic acid, and
(B) 50/50 methanol and 2 mM ammonium acetate in 0.1% formic acid. Elution of the
analytes through a Waters OASIS HLB 2.1 x 20 mm x 5 μm column (Wexford,
Ireland) was effected by an initial gradient flow of 0.6 mL/min 50% A and 50% B, then
ramped to 100% B over 0.8 min, followed by 0.1 min reconditioning. In ESI+ mode,
the MRM protonated adduct transitions m/z 315.2 to 220.2 and m/z 371.1 to 234.2 for
valdecoxib and parecoxib respectively, and m/z 318.2 to 222.2 for valdecoxib-13C215N
were monitored. Capillary voltage was set at 1.2 kV for both analytes, while cone
voltages of 30 V and 35 V, and collision energies of 25 eV and 20 eV were used for
valdecoxib and parecoxib respectively. The source temperature was 400oC for both
analytes. Under these conditions, the retention times for valdecoxib and parecoxib,
its labelled internal standard, were 1.1 and 1.3 min respectively. In plasma, linearity
was established for both valdecoxib and parecoxib over the concentration range of 10
g/L to 1869 g/L (r2 = 0.999) and 9 g/L to 2010 g/L (r2 = 0.999) respectively. In
milk, linearity was also established for both valdecoxib and parecoxib over the
concentration range of 1 mcg/L to 201 g/L (r2 = 0.999) and 0.9 g/L to 217 g/L (r2 =
0.999) respectively.
Matrix effects, absolute recovery, process efficiency and inter-patient (intra-day)
variability (relative standard deviation; RSD) for the assay were investigated as
previously described34 using 5 blank plasma and milk samples from different patients.
For sample set 1, plasma was spiked with parecoxib at 10 g/L, 100 g/L and 2000
g/L, and with valdecoxib at 10 g/L, 100 g/L and 1000 g/L and milk was spiked
with parecoxib at 1 g/L, 10 g/L and 100 g/L, and with valdecoxib at 1 g/L, 10
g/L and 100 g/L. Internal standard valdecoxib-13C215N at was spiked into plasma at
100 g/L and milk at 2 g/L. For sample set 2 spiked post-extraction addition extracts
for the same 5 samples were also prepared at concentrations equivalent to those
above by using a fixed volume of the extracts obtained after the methanolprecipitation (plasma), or after the dichloromethane extraction and reconstitution in
methanol (milk) steps. Finally, for sample set 3 “pure solutions” of all analytes at
concentrations equivalent to those described above were prepared by diluting with
methanol.
These data are summarized in the Appendix Tables 1 and 2 (see
Supplemental Digital Content 3, http://links.lww.com/AA/A353). Additional intra-day
RSD data (n=5) for parecoxib gave values of 6.5% at 1 g/L in plasma and 5.7% at
1.5 g/L in milk.
For plasma, the inter-day RSD at 200 g/L, 400 g/L and 800 g/L (n=5) were 3.3%,
5.2% and 1.7% respectively for parecoxib, and 2.3%, 1.9% and 1.1% respectively for
valdecoxib. For milk, the inter-day RSD at 10 g/L and 100 g/L (n=5) were 10.2%
and 2.0% respectively for parecoxib and 7.2% and 2.2% respectively for valdecoxib.
Limits of quantitation for the assay were set at 1.5g/L and 1 g/L for parecoxib in
milk and plasma respectively and at 10g/L for valdecoxib in both milk and plasma.