Validation of an analytical LC-MS/MS method in human plasma for the pharmacokinetic study of atomoxetine

This study aimed to validate a sensitive and reliable analytical method for the pharmacokinetic study of atomoxetine in human plasma by liquid chromatography-electrospray ionization-tandem mass spectrometry. Metoprolol was used as an internal standard. After liquid-liquid extraction with methyl t-butyl ether, the supernatant was evaporated. The residue was then reconstituted and an aliquot was injected into the high performance liquid chromatographic system. Separation was performed on a Phenomenex Luna C₁₈ column (2.0 mm × 100 mm, 3 μm particles) with a mobile phase of 10 mM ammonium formate buffer: methanol = 10: 90 (v/v). Tandem mass spectrometry was performed in the electrospray ionization positive ion mode using the multiple reaction monitoring mode for quantification. The mass transition pairs of m/z 256 → 44 for atomoxetine and m/z 268 → 116 for the internal standard were used. The flow rate of the mobile phase was 0.25 mL/min and the retention times of atomoxetine and the internal standard were found to be 1.0 and 0.9 min, respectively. The calibration curve for atomoxetine was linear in the concentration range of 1–750 ng/mL (r ² = 0.9992) with a lower limit of quantification of 1 ng/mL. The mean accuracy for atomoxetine was 93–102%. The coefficients of variation (precision) in the intra- and inter-day validation for atomoxetine were 4.0–6.8 and 1.1–9.6%, respectively. The pharmacokinetic parameters of atomoxetine were evaluated after administration of a 40-mg single oral dose to twelve healthy male volunteers. The mean AUC_{0–24 h}, C _max, T _max and T _1/2 for atomoxetine were 1.9 ± 0.8 μg h/mL, 0.34 ± 0.11 μg/mL, 1.0 ± 0.5 h and 3.9 ± 1.3 h, respectively.     

Validation of an LC-MS/MS method for the quantitative determination of a novel fungicide: pyraoxystrobin in rat plasma and its application to a toxicokinetics study

A simple, sensitive, and reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of pyraoxystrobin in rat plasma and applied to a toxicokinetics study. The separation was performed by gradient elution on a Luna 5 μ C18 (2) 100 Å column (50?×?4.6 mm I.D., 5 μm) with mobile phase: water (0.1 % formic acid, v/v)/acetonitrile (0.1 % formic acid, v/v), followed by quantification with a mass detector in multiple reaction monitoring (MRM) mode using ESI as an interface. The calibration curve was linear over a concentration range of 1.00–200 ng/mL. The recovery for pyraoxystrobin ranged from 101.4 to 108.2 %. The intraday bias and precision ranged from ?9.3 to 8.1 % and from 0.7 to 8.4 %, respectively, and the interday bias and precision ranged from ?0.3 to 4.0 % and from 4.4 to 7.2 %, respectively. The toxicokinetics of pyraoxystrobin after single 100 and 1,000 mg/kg oral doses were studied in rats. Figure

The chromatogram of pyraoxystrobin highest calibration standard (ULOQ) extract.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Dried plasma spots as an alternative sample collection technique for the quantitative LC-MS/MS determination of gabapentin

Dried plasma spots were employed as an alternative sample collection technique for the quantitative determination of gabapentin in human plasma, using an automated liquid chromatography tandem mass spectrometry method. After the methanolic extraction of single plasma, 1/8-in. disks which contained only 1.98 μL plasma volume, were placed in 96-well format plates, then gabapentin and its internal standard, 4-aminocyclohexanecarboxylic acid, were derivatized with n-butanol/HCl (3 M) and detected as butyl esters. The assay exhibited excellent linearity over the concentration range of 40.0–10.0 × 10³ ng/mL, which is suitable for the determination of gabapentin after per os administration of a single tablet. Variations in intra- and inter-assay accuracy and precision were within internationally accepted criteria. Homogeneity of plasma spots was proven, whilst butyl ester stability for both analytes was estimated and found very satisfactory. The quantitative analysis of Gabapentin with dried plasma spot specimens seems to be a prominent and advantageous technique, especially when applied to pharmacokinetic studies, where plasma sampling procedure becomes rapid and required plasma volumes negligible.     

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.     

Development and validation of a LC-MS/MS method for d-cycloserine determination in human plasma for bioequivalence study

A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-d -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150?×?4.6 mm; 3 μm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103–75?m/z for cycloserine and 124 to 80?m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3–30 μg/mL was established for cycloserine using 0.2 mL human plasma and a 1 μL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8–3.4 %; accuracy, 93.8–104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.     

LC-MS/MS method for the determination of cynandione A in rat plasma and tissues

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the quantitative determination of cynandione A in rat plasma and tissues. The plasma samples were pretreated by liquid-liquid extraction with ethyl acetate after the internal standard (honokiol) had been spiked. The tissue samples were homogenized with physiological saline and treated further like the plasma samples. The separation was performed using a Zorbax SB-C(18) column (3.5 microm, 2.1 x 100 mm) and a C18 guard column (5 microm, 4.0 x 2.0 mm) with an isocratic mobile phase consisting of methanol-0.1% formic acid (78:22, v/v) at a flow rate of 0.2 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple-reaction monitoring mode using the electrospray ionization technique in negative mode. The nominal retention times for cynandione A and honokiol were 1.41 and 2.63 min, respectively. The method was validated within the concentration range 0.2-1000 ng/mL in plasma and homogenized tissue for cynandione A, and the calibration curves were linear with correlation coefficients >0.992. The lower limit of quantification of cynandione A was 0.2 ng/mL. The intra-day and inter-day precision and accuracy of the assay in plasma were less than 14.4%, while the intra-day and inter-day precision and accuracy of the assay in tissue homogenate were less than 14.2%. This method proved to be suitable for study of pharmacokinetics and tissue distribution of cynandione A in rat.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

Validation of a multi-residue method for the determination of several antibiotic groups in honey by LC-MS/MS

The presented multi-method was developed for the confirmation of 37 antibiotic substances from the six antibiotic groups: macrolides, lincosamides, quinolones, tetracyclines, pleuromutilines and diamino-pyrimidine derivatives. All substances were analysed simultaneously in a single analytical run with the same procedure, including an extraction with buffer, a clean-up by solid-phase extraction, and the measurement by liquid chromatography tandem mass spectrometry in ESI+ mode. The method was validated on the basis of an in-house validation concept with factorial design by combination of seven factors to check the robustness in a concentration range of 5-50 μg kg(-1). The honeys used were of different types with regard to colour and origin. The values calculated for the validation parameters-decision limit CCα (range, 7.5-12.9 μg kg(-1)), detection capability CCβ (range, 9.4-19.9 μg kg(-1)), within-laboratory reproducibility RSD(wR) (<20% except for tulathromycin with 23.5% and tylvalosin with 21.4 %), repeatability RSD(r) (<20% except for tylvalosin with 21.1%), and recovery (range, 92-106%)-were acceptable and in agreement with the criteria of Commission Decision 2002/657/EC. The validation results showed that the method was applicable for the residue analysis of antibiotics in honey to substances with and without recommended concentrations, although some changes had been tested during validation to determine the robustness of the method.     

Validation of an LC/MS method for the determination of gemfibrozil in human plasma and its application to a pharmacokinetic study

Gemfibrozil, a fibric acid hypolipidemic agent, is increasingly being used in clinical drug–drug interaction studies as an inhibitor of drug metabolizing enzymes and drug transporters. The validation of a fast, accurate and precise LC/MS method is described for the quantitative determination of gemfibrozil in an EDTA‐anticoagulated human plasma matrix. Briefly, gemfibrozil was extracted from human plasma by an acetonitrile protein precipitation method. The assay was reproducible with intra‐assay precision between 1.6 and 10.7%, and inter‐assay precision ranging from 4.4 to 7.8%. The assay also showed good accuracy, with intra‐assay concentrations within 85.6–108.7% of the expected value, and inter‐assay concentrations within 89.4–104.0% of the expected value. The linear concentration range was between 0.5 and 50 µg/mL with a lower limit of quantitation of 0.5 µg/mL when 125 µL of plasma were extracted. This LC/MS method yielded a quick, simple and reliable protocol for determining gemfibrozil concentrations in plasma and is applicable to clinical pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.     

A sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide in human plasma and pharmacokinetic studies

Pomalidomide is an immunomodulatory agent （IMiD） that has been approved by the US Food and Drug Administration （FDA） for clinical treatment of patients with multiple myeloma.In this work,we developed a sensitive and validated LC-MS/MS method for high-throughput determination of pomalidomide over the range of 1.006–100.6 ng/m L （R²=0.9991） in human plasma and pharmacokinetic studies.A liquid-liquid extraction method using ethyl acetate was applied to extract pomalidomide and afatinib （...     

Validation of a sensitive LC/MS/MS method for the determination of telaprevir and its R‐isomer in human plasma

The purpose of this study was to validate a reversed‐phase high‐performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R‐diastereomer (VRT‐127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir‐R was performed on a Waters XBridge^TM BEH Shield C₁₈, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir‐d11). An internal standard for the telaprevir‐R (telaprevir‐R‐d11) was also prepared by incubating telaprevir‐d11 in basic solution, which facilitated isomer inter‐conversion. The detection and quantitation of telaprevir, telaprevir‐R, telaprevir‐IS and telaprevir‐R‐IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography–tandem mass spectrometry (LC/MS/MS) with liquid–liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra‐ and inter‐batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.     

Development and validation of an HPLC/MS/MS method for the determination of sufentanil and morphine in human plasma

A sensitive and fast HPLC/MS/MS method for measurement of sufentanil and morphine in plasma was developed and validated. A single liquid-liquid extraction in alkaline medium was used for the cleanup of plasma, and fentanyl was added as an internal standard (IS). The analyses were carried out using a C18 column and the mobile phase acetonitrile-5 mM ammonium acetate + 0.25% formic acid (70 + 30, v/v). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect precursor --> product ion transition 387.0 > 238.0, 285.7 > 165.1, and 337.0 > 188.0 for sufentanil, morphine, and IS, respectively. The method was linear in the 0.05 (LOQ) - 500 ng/mL range for sufentanil and 10 (LOQ) - 1000 ng/mL range for morphine. Good selectivity, linearity, precision, accuracy, and robustness were obtained for the HPLC/MS/MS method. The proposed method was successfully applied for the determination of sufentanil and morphine in patients undergoing cardiac surgery.     

Development and validation of LC-MS/MS method for the quantification of oxcarbazepine in human plasma using an experimental design

A rapid tandem mass spectrometric (MS-MS) method for the quantification of Oxcarbazepine (OXB) in human plasma using imipramine as an internal standard (IS) has been developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 3.0 min, using a mobile phase of acetonitrile-10 mM ammonium formate (90 : 10 v/v) at a flow rate of 0.3 ml/min. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 253>208 and m/z 281>86 for OXB and the IS respectively. Calibration curves were linear over the concentration range of 0.2-16 mug/ml (r>0.999) with a limit of quantification of 0.2 mug/ml. Analytical recoveries of OXB from spiked human plasma were in the range of 74.9 to 76.3%. Plackett-Burman design was applied for screening of chromatographic and mass spectrometric factors; factorial design was applied for optimization of essential factors for the robustness study. A linear model was postulated and a 2(3) full factorial design was employed to estimate the model coefficients for intermediate precision. More specifically, experimental design helps the researcher to verify if changes in factor values produce a statistically significant variation of the observed response. The strategy is most effective if statistical design is used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic and bioequivalence studies.     

Validation and application of an LC-MS/MS method for the simultaneous quantification of 13 pharmaceuticals in seawater

Knowledge of the presence of micropollutants such as pharmaceuticals, in coastal areas, is very limited; therefore, the main objective of this study was to optimize and validate a new analytical method for the quantitative analysis of 13 multiclass pharmaceuticals in seawater. Target compounds included antibiotics, non-steroidal anti-inflammatory drugs, β-blockers, lipid regulators and one psychiatric drug. A combination of solid-phase extraction and liquid chromatography coupled with multiple mass spectrometry enabled their detection at the low nanogram per litre level. The limits of quantification varied between 1 and 50 ng L^-1, for most components the linearities were more than 0.99 and the recoveries obtained in seawater (95–108%) were satisfactory. This method was applied to seawater and estuarine water samples collected in the Belgian coastal zone, to assess the prevalence of common pharmaceuticals in this marine environment. Seven pharmaceuticals, including compounds of which the presence in marine environments had not been reported earlier, were detected, with salicylic acid and carbamazepine being the most abundant, in concentrations up to 855 ng L^-1.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of amlodipine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of amlodipine in human plasma. Amlodipine was extracted from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at sub-nanogram levels. The proposed method has been validated for a linear range of 0.1-10.0 ng/mL with correlation coefficient >or=0.9990. The intrarun and interrun precision and accuracy were within 10.0%. The overall recovery for amlodipine was 63.67%. Total run time was 3.2 min only.     

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-β fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.