Quantification of the Aminosteroidal Non ... - Clinical Chemistry

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Daly AK, Brockmöller J, Broly F, Eichelbaum M, Evans WE, Gonzalez FJ, et al. ... debrisoquine: characterization and PCR-based detection of alleles with.

Clinical Chemistry 46, No. 9, 2000

References 1. Brøsen K, Gram LF. Clinical significance of the sparteine/debrisoquine oxidation polymorphism. Eur J Clin Pharmacol 1989;36:537– 47. 2. Kimura S, Umeno M, Skoda RC, Meyer UA, Gonzalez F. The human debrisoquine 4-hydroxylase (CYP2D6) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene and a pseudogene. Am J Hum Genet 1989;45:889 –904. 3. Daly AK, Brockmo¨ller J, Broly F, Eichelbaum M, Evans WE, Gonzalez FJ, et al. Nomenclature for human CYP2D6 alleles. Pharmacogenetics 1996;6:193– 201. 4. Bertilsson L. Geographical/interracial differences in polymorphic drug oxidation [Review]. Clin Pharmacokinet 1995;29:192–209. 5. Bertilsson L, Dahl ML, Tybring G. Pharmacogenetics of antidepressants: clinical aspects. Acta Psychiatr Scand Suppl 1997;391:14 –21. 6. Johansson I, Lundqvist E, Bertilsson L, Dahl M-L, Sjo¨qvist F, IngelmanSundberg M. Inherited amplification of an active gene in the cytochrome P450 CYP2D6 locus as a cause of ultrarapid metabolism of debrisoquine. Proc Natl Acad Sci U S A 1993;90:11825–9. 7. Aklillu E, Persson I, Bertilsson L, Johansson I, Rodrigues F, IngelmanSundberg M. Frequent distribution of ultrarapid metabolizers of debrisoquine in an Ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles. J Pharmacol Exp Ther 1996;278:441– 6. 8. McLellan RA, Oscarsson M, Seide Gård J, Price Evans DA, IngelmanSundberg M. Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians. Pharmacogenetics 1997;7:187–91. 9. Agundez J, Ledesma M, Ladero J, Benitez J. Prevalence of CYP2D6 gene duplication and its repercussion on the oxidative phenotype in a white population. Clin Pharmacol Ther 1994;57:265–9. 10. Ingelman-Sundberg M. Duplication, multiduplication, and amplification of genes encoding drug-metabolizing enzymes: evolutionary, toxicological, and clinical pharmacological aspects [Review]. Drug Metab Rev 1999;31:449 – 59. 11. Johansson I, Oscarsson M, Yue Q-Y, Bertilsson L, Sjo¨qvist F, IngelmanSundberg M. Genetic analysis of the Chinese cytochrome P450 locus: characterization of variant CYP2D6 genes present in subjects with diminished capacity for debrisoquine hydroxylation. Mol Pharmacol 1994;46: 452–9. 12. Dahl M-L, Yue Q-Y, Roh H-K, Johansson I, Sa¨we J, Sjo¨qvist F, Bertilsson L. Genetic analysis of the CYP2D6 locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects. Pharmacogenetics 1995;5:159 – 64. 13. Wang SL, Huang JD, Lai MD, Liu BH, Lai ML. Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: polymorphism in RFLP and DNA sequence of CYP2D6. Clin Pharmacol Ther 1993;53:410 – 8. 14. Yokoi T, Kosaka Y, Chida M, Chiba K, Nakamura H, Ishizaki T, et al. A new CYP2D6 allele with a nine base insertion in exon 9 in a Japanese population associated with poor metabolizer phenotype. Pharmacogenetics 1996;5: 395– 441. 15. Garcia-Barcelo´ M, Chow LY, Chiu HFK, Wing YK, Lee DTS, Lam KL, Waye MMY. Genetic analysis of the CYP2D6 locus in a Hong Kong Chinese population. Clin Chem 2000;46:18 –23. 16. Løvlie R, Daly K, Molven A, Idle JR, Steen VM. Ultrarapid metabolizers of debrisoquine: characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene. FEBS Lett 1996;392:30 – 4. 17. Lundqvist E, Johansson I, Ingelman-Sundberg M. Genetic mechanisms for duplication and multiduplication of the human CYP2D6 gene and methods for detection of duplicated CYP2D6 genes. Gene 1999;226:327–38. 18. Heim HH, Meyer UA. Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6. Genomics 1992;14:49 –58. 19. Bertilsson L, Dahl M-L, Sjo¨qvist F, Åberg-Wistedt A, Humble M, Johansson I, et al. Molecular basis for rational megaprescribing in ultrarapid hydroxylators of debrisoquine [Letter]. Lancet 1993;341:63. 20. Masimirembwa CM, Johansson I, Hasler JA, Ingelman-Sundberg M. Genetic polymorphism of cytochrome P450 CYP2D6 in Zimbabwean population. Pharmacogenetics 1993;3:275– 80. 21. Johansson I, Yue QY, Dahl M-L, Heim M, Sa¨we J, Bertilsson L, et al. Genetic analysis of the interethnic difference between Chinese and Caucasians in the polymorphic metabolism of debrisoquine and codeine. Eur J Clin Pharmacol 1991;40:553– 6.

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Quantification of the Aminosteroidal Non-Depolarizing Neuromuscular Blocking Agents Rocuronium and Vecuronium in Plasma with Liquid Chromatography-Tandem Mass Spectroscopy, Ursula Gutteck-Amsler and Katharina M. Rentsch* (Institute of Clinical Chemistry, University Hospital Zu¨rich, Ra¨mistrasse 100, CH-8091 Zu¨rich, Switzerland; * author for correspondence: fax 411-255-4590, e-mail [email protected]) Rocuronium (RO) and vecuronium (VE) are widely used aminosteroidal non-depolarizing neuromuscular blocking agents. There are few methods published for the determination of VE, its metabolite 3-desacetyl-vecuronium (OHV) (1– 4 ), and RO (5, 6 ) that use HPLC with ultraviolet, fluorescence, or electrochemical detection or gas chromatography with nitrogen-sensitive detection. To date, the methods published for the determination of VE and OHV use an exotic detection system (1 ), a timeconsuming derivatization step (3 ), or a very laborious analytical technique (2, 4 ). The published methods for RO need a very sophisticated instrument that requires postseparation extraction of the drugs (6 ) or include a very time-consuming derivatization step (5 ). Despite the great analytical effort, the reproducibility (CV ⬎10%) and detection limits (⬎10 ␮g/L) of these methods are not satisfying. To perform pharmacokinetic studies of RO, VE, and its metabolite OHV, we have established a robust, sensitive, and specific liquid chromatography electrospray ionization-tandem mass spectrometry method. Immediately after blood collection into heparin-containing tubes and centrifugation at 4 °C, 1 mL of plasma was added to a tube containing 0.2 mL of a 1 mol/L sodium hydrogen phosphate solution to inhibit the degradation of the drugs. The samples were kept frozen at ⫺70 °C until analysis. Before extraction of the plasma samples, 0.25 mL of a 1 mol/L sodium hydrogen phosphate solution and 50 ␮L of the internal standard pancuronium (PA; 10 mg/L) were added to 0.5 mL of plasma. After the addition of 1 mL of 6 mol/L potassium iodide, the drugs were extracted with 5 mL of toluene on a horizontal shaker (Infors HAT; Infors) for 20 min. After centrifugation for 5 min at 1000g, the organic layer was separated and dried by evaporation (Rotavapor; Bu¨chi), and the residue was dissolved in 100 ␮L of mobile phase. The HPLC system consisted of a RHEOS 2000 pump (Flux Instruments AG), an A200S autosampler (CTC) and a LCQ ion trap mass spectrometer (Thermoquest). The ionization mode was positive electrospray with a spray voltage of 3.8 kV and a capillary temperature of 230 °C. PA was detected by the most intense product ion of its divalent cation (m/z 286.43236.7). RO was detected by the most intensive product ion of its monovalent cation (m/z 529.43487.4). VE and OHV were detected by the most intensive product ions of their protonated molecules (divalent cations; VE, m/z 279.23249.4; OHV, m/z 258.53100.2). The different neuromuscular blocking agents were separated using a Nucleosil C18 HD column (12.5 cm ⫻ 4 mm; 5 ␮m particle size; Macherey-Nagel) protected with a

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Technical Briefs

Nucleosil C18 HD guard column (8 ⫻ 4 mm; 5 ␮m particle size; Macherey-Nagel). The mobile phase consisted of 50 mmol/L ammonium formate buffer (pH 3):methanol (73: 27, by volume). The flow rate of the mobile phase was set at 0.8 mL/min, and a postcolumn flow-splitting device was used to send sample through the mass spectrometric detector at a flow rate of 100 ␮L/min. The retention times were 6.4 min for RO, 8.2 min for PA, 9.7 min for OHV, and 15.4 min for VE. Examples of chromatograms of patients’ plasma samples containing RO and VE are shown in Fig. 1, A and B, respectively. Seven calibrators were prepared by adding to human plasma corresponding amounts of RO or VE and OHV at concentrations of 0, 20, 100, 200, 500, 1000, and 2000 ␮g/L, respectively. The calibration curves were obtained by plotting the peak-area ratio for the m/z 487.4 (RO), 249.4 (VE), 100.2 (OHV), 236.7 (PA) ion pairs vs the concentration. Calibration curves were analyzed by unweighted least-squares linear regression analysis and were linear over the range studied. The precision of the calibration curves is shown in Table 1. Within-run imprecision was determined for a series of six plasma samples supplemented with each compound; the CVs (mean concentration) were 2.9% (100 ␮g/L) and 5.3% (2000 ␮g/L) for RO, 9.0% (75 ␮g/L) and 1.0% (750 ␮g/L) for VE, and 10% (75 ␮g/L) and 1.0% (750 ␮g/L) for

Table 1. Least-squares regression dataa for RO, VE, and OHV (n ⴝ 5). Drug

Slope, (␮g/L)ⴚ1

Intercept

Correlation coefficient

RO 0.0038 ⫾ 0.0002 ⫺0.0094 ⫾ 0.0100 0.9999 ⫾ 0.0001 VE 0.0028 ⫾ 0.0004 0.0507 ⫾ 0.0438 0.9994 ⫾ 0.0003 OHV 0.00054 ⫾ 0.00013 0.0046 ⫾ 0.0108 0.9990 ⫾ 0.0004 a

All values are the mean ⫾ SD.

OHV. The between-run CVs were 7.4% (50 ␮g/L) and 5.3% (750 ␮g/L) for RO, 7.2% (75 ␮g/L) and 2.7% (750 ␮g/L) for VE, and 15% (75 ␮g/L) and 4.0% (750 ␮g/L) for OHV. The mean accuracy of the presented method was 106% for RO, 90% for VE, and 89% for OHV. To determine the extraction recovery of the method, different plasma samples to which RO, VE, and OHV had been added were analyzed and compared with calibrators in mobile phase containing the same amount of drug. The recovery of added RO was 66%. The recoveries for VE and OHV were 78% and 70%, respectively. The limit of quantification for the method was calculated using a signal-to-noise ratio of 3. For this purpose, the noise signal was obtained as the amplitude of the peaks from a segment of the chromatogram that preceded each peak. The quantification limits were 5 ␮g/L for RO and 15 ␮g/L for VE and OHV. In conclusion, the method described met all of the analytical requirements necessary to analyze a large series of patient samples, e.g., pharmacokinetic studies. The reproducibility and sensitivity for the determination of RO was markedly improved compared with previously published analytical techniques. This method was applied to three kinetic studies in humans and to one study in rats.

We gratefully acknowledge Rene´ Bu¨hrer for excellent technical assistance. References

Fig. 1. Chromatograms of patients’ plasma samples containing RO and PA (A) and VE, OHV, and PA (B). PA was added as the internal standard.

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