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.     

The chiral bioconversion and preclinical pharmacokinetic analysis of (R)‐(+)‐rabeprazole in beagle dogs by HPLC and HPLC‐MS/MS

In order to accurately investigate the preclinical pharmacokinetics of (R)‐(+)‐rabeprazole sodium injection, a reliable high‐performance liquid chromatography (HPLC) method was developed using a Chiral‐AGP column to prove that there is no chiral bioconversion of (R)‐(+)‐rabeprazole to (S)‐(?)‐rabeprazole in beagle dogs after single intravenous administration of (R)‐(+)‐rabeprazole sodium injection. An HPLC–tandem mass spectrometry (HPLC‐MS/MS) method for analysis of (R)‐(+)‐rabeprazole was developed and validated, and used to acquire the pharmacokinetic parameters in beagle dogs. (R)‐(+)‐Rabeprazole and internal standard omeprazole were extracted from plasma samples by protein precipitation and separated on a C₁₈ column using methanol–5 mm ammonium acetate as mobile phase. Detection was performed using a turbo‐spray ionization source and mass spectrometric positive multi‐reaction monitoring mode. The linear relationship was achieved in the range from 2.5 to 5000 ng/mL. The method also afforded satisfactory results in terms of sensitivity, specificity, precision, accuracy and recovery as well as the stability of the analyte under various conditions, and was successfully applied to a preclinical pharmacokinetic study in beagle dogs after single intravenous administrations of (R)‐(+)‐rabeprazole sodium injection at 0.33, 2 and 6 mg/kg. Copyright © 2013 John Wiley & Sons, Ltd.     

Enantioselective determination of R‐(−)‐manidipine and S‐(+)‐manidipine in human plasma by a sensitive and selective chiral LC–MS/MS assay

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and validated for the enantioselective determination of manidipine in human plasma using isotope‐labeled compounds as internal standards. After solid‐phase extraction, R ‐(−)‐manidipine and S ‐(+)‐manidipine were chromatographed on a Chiralpack IC‐3 C₁₈ column using a isocratic mobile phase composed of 2 mm ammonium bicarbonate and acetonitrile (15:85, v /v). The precursor ion to product ion transitions for the enantiomers and internal standards were monitored in the multiple reaction monitoring and positive ionization mode using an API‐4000 mass spectrometer. The method was linear over the concentration range of 0.05–10.2 ng/mL for both enantiomers. The precision and accuracy results over five concentration levels in five different batches were well within the acceptance limits. The mean extraction recovery was >80% for both enantiomers. A variety of stability tests were executed in plasma and in neat samples, which complies with the FDA guidelines. After complete validation, the method was successfully applied to a pharmacokinetic study of a manidipine 20 mg oral dose in 10 healthy South India subjects under fasting conditions. The assay reproducibility is shown through incurred samples reanalysis of 20 subject plasma samples.     

Novel UHPLC‐MS/MS method for the determination of rotigotine in the plasma of patients with Parkinson's disease

A novel ultra‐high‐pressure liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid–liquid extraction with tert‐ butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate–5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra‐ and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinson's disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

Doping control analysis of desmopressin in human urine by LC‐ESI‐MS/MS after urine delipidation

The World Anti‐Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high‐performance liquid chromatography–electrospray tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di‐isopropyl ether/n‐butanol and solid‐phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.     

An improved LC‐MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and its active metabolites in human plasma and its application to a pharmacokinetic study in patients

In this study, a sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry method for the simultaneous determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and its active metabolites, 1‐caffeoyl‐5‐feruoylquinic acid and 1,5‐O‐diferuoylquinic acid, in human plasma, using puerarin as internal standard, was developed and validated. Analytes were extracted from plasma samples by liquid–liquid extraction with ethyl acetate, separated on a C₁₈ reversed‐phase column with water containing 5 mM ammonium acetate and acetonitrile as the mobile phase and detected by electrospray ionization mass spectrometry in negative selected reaction monitoring mode. The accuracy and precision of the method were acceptable and linearity was good over the range 1–200 ng/mL for each analyte. In addition, the selectivity, extraction recovery and matrix effect were satisfactory too. The validated LC‐MS/MS method was successfully applied to phase II clinical pharmacokinetic study of 1,5‐DCQA in patients. Copyright © 2010 John Wiley & Sons, Ltd.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Quantitative determination of periplocymarin in rat plasma and tissue by LC‐MS/MS: application to pharmacokinetic and tissue distribution study

A simple, rapid and sensitive liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method for the determination of periplocymarin in biological samples was developed and successfully applied to the pharmacokinetic and tissue distribution study of periplocymarin after oral administration of periplocin. Biological samples were processed with ethyl acetate by liquid–liquid extraction, and diazepam was used as the internal standard. Periplocymarin was analyzed on a C₁₈ column with isocratic eluted mobile phase composed of methanol and water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min (73:27, v/v). Detection was performed on a triple‐quadrupole tandem mass spectrometer using positive‐ion mode electrospray ionization in the selected reaction monitoring mode. The MS/MS ion transitions monitored were m/z 535.3→355.1 and 285.1→193.0 for periplocymarin and diazepam, respectively. Good linearity was observed over the concentration ranges. The lower limit of quantification was 0.5 ng/mL in plasma and tested tissues. The intra‐and inter‐day precisions (relative standard deviation) were <10.2 and 10.5%, respectively, and accuracies (relative error) were between ?6.8 and 8.9%. Recoveries in plasma and tissue were >90%. The validated method was successfully applied to the pharmacokinetic and tissue distribution studies of periplocymarin in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid simultaneous determination of codeine and morphine in plasma using LC‐ESI‐MS/MS: Application to a clinical pharmacokinetic study

A rapid and sensitive high‐performance LC‐MS/MS method was developed and validated for the simultaneous quantification of codeine and its metabolite morphine in human plasma using donepezil as an internal standard (IS). Following a single liquid‐liquid extraction with ethyl acetate, the analytes were separated using an isocratic mobile phase on a C₁₈ column and analyzed by MS/MS in the selected reaction monitoring mode using the respective [M+H]⁺ ions, mass‐to‐charge ratio (m/z) 300/165 for codeine, m/z 286/165 for morphine and m/z 380/91 for IS. The method exhibited a linear dynamic range of 0.2–100/0.5–250 ng/mL for codeine/morphine in human plasma, respectively. The lower LOQs were 0.2 and 0.5 ng/mL for codeine and its metabolite morphine using 0.5 mL of human plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated LC‐MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 30 mg codeine phosphate.     

A rapid UPLC‐MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics

A reliable high‐throughput ultra‐high performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for oleanolic acid (OA) determination in rat plasma and liver tissue using glycyrrhetic acid as the internal standard (IS). Plasma and liver homogenate samples were prepared using solid‐phase extraction. Chromatographic separation was achieved on a C₁₈ column using an isocratic mobile phase system. The detection was performed by multiple reaction monitoring mode via positive electrospray ionization interface. The calibration curves showed good linearity (R² > 0.9997) within the tested concentration ranges. The lower limit of quantification for plasma and liver tissue was ≤0.75 ng/mL. The intra‐ and inter‐day precision and accuracy deviations were within ±15% in plasma and liver tissue. The mean extraction recoveries ranged from 80.8 to 87.0%. In addition, the carryover, matrix effect, stability and robustness involved in the method were also validated. The method was successfully applied to the plasma and hepatic pharmacokinetics of OA after oral administration to rats. Copyright © 2015 John Wiley & Sons, Ltd.     

LC‐MS/MS determination and pharmacokinetic study of lacidipine in human plasma

A robust, specific and fully validated LC‐MS/MS method as per general practices of industry has been developed for estimation of lacidipine (LAC) with 100 μL of human plasma using lacidipine‐¹³C8 as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode. A simple liquid–liquid extraction process was used to extract LAC and IS from human plasma. The total run time was 3.0 min and the elution of LAC and IS occurred at 1.96 and 1.97 min; this was achieved with a mobile phase consisting of 5 mm ammonium acetate buffer–acetontrile (15:85 v/v) at a flow rate of 0.60 mL/min on a Zorbax SB C₁₈ (50 × 4.6 mm, 5 µm) column. A linear response function was established for the range of concentrations 50–15,000 pg/mL (r > 0.998) for LAC. The current developed method has negligible matrix effect and is free from unwanted adducts and clusters which are formed owing to system such as solvent or mobile phase. The developed assay method was applied to an oral pharmacokinetic study in humans and successfully characterized the pharmacokinetic data up to 72 h. Copyright © 2013 John Wiley & Sons, Ltd.     

LC‐MS/MS assay for the determination of lurasidone and its active metabolite,ID‐14283 in human plasma and its application to a clinical pharmacokinetic study

The authors proposed a sensitive, selective and rapid liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay procedure for the quantification of lurasidone and its active metabolite, i.e. ID‐14283 in human plasma simultaneously using corresponding isotope labeled compounds as internal standards as per regulatory guidelines. After liquid–liquid extraction with tert‐butyl methyl ether, the analytes were chromatographed on a C₁₈ column using an optimized mobile phase composed of 5 mm ammonium acetate (pH 5.0) and acetonitrile (15:85, v/v) and delivered at a flow rate of 1.00 mL/min. The assay exhibits excellent linearity in the concentration ranges of 0.25–100 and 0.10–14.1 ng/mL for lurasidone and ID‐14283, respectively. The precision and accuracy results over five concentration levels in four different batches were well within the acceptance limits. Lurasidone and ID‐14283 were found to be stable in battery of stability studies. The method was rapid with the chromatographic run time 2.5 min, which made it possible to analyze 300 samples in a single day. Additionally, this method was successfully used to estimate the in vivo plasma concentrations of lurasidone and ID‐14283 obtained from a pharmacokinetic study in south Indian male subjects and the results were authenticated by conducting incurred samples reanalysis. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of aurantio‐obtusin,chrysoobtusin, obtusin and 1‐desmethylobtusin in rat plasma by UHPLC‐MS/MS

A sensitive and reliable ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for the simultaneous determination of four active components of Semen Cassiae extract (aurantio‐obtusin, chrysoobtusin, obtusin and 1‐desmethylobtusin) in rat plasma after oral administration. Chromatographic separation was achieved on an Agilent Poroshell 120 C₁₈ column with gradient elution using a mobile phase that consisted of acetonitrile‐ammonium acetate in water (30 mm ) at a flow rate of 0.4 mL/min. Detection was performed by a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The calibration curve was linear over a range of 3.24–1296 ng/mL for aurantio‐obtusin, 0.77–618 ng/mL for chrysoobtusin, 34.55–1818 ng/mL for obtusin and 1.86–1485 ng/mL for 1‐desmethylobtusin. Inter‐ and intra‐day assay variation was <15%. All analytes were shown to be stable during all sample storage and analysis procedures. Copyright © 2013 John Wiley & Sons, Ltd.     

A UPLC‐MS/MS method for simultaneous determination of five flavonoids from Stellera chamaejasme L. in rat plasma and its application to a pharmacokinetic study

Stellera chamaejasme L. has been used as a traditional Chinese medicine for the treatment of scabies, tinea, stubborn skin ulcers, chronic tracheitis, cancer and tuberculosis. A sensitive and selective ultra‐high liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method was developed and validated for the simultaneous determination of five flavonoids (stelleranol, chamaechromone, neochamaejasmin A, chamaejasmine and isochamaejasmin) of S. chamaejasme L. in rat plasma. Chromatographic separation was accomplished on an Agilent Poroshell 120 EC‐C₁₈ column (2.1 × 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min and the total analysis time was 7 min. The analytes were detected using multiple reaction monitoring in positive ionization mode. The samples were prepared by liquid–liquid extraction with ethyl acetate. The UPLC‐MS/MS method was validated for specificity, linearity, sensitivity, accuracy and precision, recovery, matrix effect and stability. The validated method exhibited good linearity (r ≥ 0.9956), and the lower limits of quantification ranged from 0.51 to 0.64 ng/mL for five flavonoids. The intra‐ and inter‐day precision were both <10.2%, and the accuracy ranged from −11.79 to 9.21%. This method was successfully applied to a pharmacokinetic study of five flavonoids in rats after oral administration of ethyl acetate extract of S. chamaejasme L.     

Development and validation of an LC‐ESI‐MS/MS method for the quantitation of hemslecin A in rhesus monkey plasma and its application in pharmacokinetics

In this study, a new LC‐ESI‐MS/MS‐based method was validated for the quantitation of hemslecin A in rhesus monkey plasma using otophylloside A as internal standard (IS). Hemslecin A and the IS were extracted from rhesus monkey plasma using liquid–liquid extraction as the sample clean‐up procedure, and were subjected to chromatography on a Phenomenex Luna CN column (150 × 2.0 mm, 3.0 µm) with the mobile phase consisting of methanol and 0.02 mol/mL ammonium acetate (55:45, v/v) at a flow rate of 0.2 mL/min. Detection was performed on an Agilent G6410B tandem mass spectrometer by positive ion electrospray ionization in multiple reaction monitoring mode, monitoring the transitions m/z 580.5 [M + NH₄]⁺ → 503.4 and m/z 518.2 [M + NH₄]⁺ → 345.0 for hemslecin A and IS, respectively. The assay was linear over the concentration range of 0.5–200 ng/mL and was successfully applied to a pharmacokinetic study in rhesus monkeys. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.