Ion Spray Interface Research Articles

Determination and stability of CP-31398 in plasma from experimental animals by LC-MS/MS

A sensitive and accurate approach for the determination of CP-31398 (N-{2-[( E)-2-(4-methoxy-phenyl)-vinyl]-quinazolin-4-yl}-N′,N′-dimethyl-propane-1,3-diamine hydrochloride) in rat and dog plasma by LC-MS/MS was validated to support preclinical toxicological and pharmacological studies. Based on the results of stability experiments with diluted CP-31398 solutions using NMR, LC-MS/MS and LC-Q-TOF, all sample preparation and handling steps were performed under yellow light to avoid CP-31398 decomposition. CP-31398 was extracted by protein precipitation with acetonitrile and separated using a Phenomenex Luna 3 μm phenyl-hexyl, 100 Å, 30 × 2.0 mm column (rat plasma) or a Phenomenex Synergi 4μ Polar-RP, 80 Å, 30 × 2.0 mm column (dog plasma) at a flow rate of 0.30 mL/min. The mobile phase consisted of A: 1% formic acid in water and B: 1% formic acid in methanol or acetonitrile. Total run times for rat and dog samples were 7 and 8 min, respectively, with accompanying retention times of 1.8 for both columns. A turbo ion spray interface was used as the ion source operating in positive mode. Calibration curves were linear from 5 to 1000 ng/mL. Linearity was assessed using the external standard method. Within-run and between-run accuracy was 93–109% of the true value for all analytes with precision (SD) of 8% or less for all experiments. The validated method was applied to preclinical toxicology studies in rats and dogs after oral administration of CP-31398.

Simultaneous quantification of flavonoids and phenolic acids in Herba Scutellariae barbatae and its confused plants by high performance liquid chromatography-tandem mass spectrometry

A sensitive and selective high performance liquid chromatography–tandem mass spectrometric method (HPLC–MS/MS) has been developed for simultaneous analysis of 11 components (7 flavonoids and 4 phenolic acids) in Herba Scutellariae barbatae. Simultaneous separation of these 11 compounds was achieved on a C18 analytical column with gradient elution of methanol and 0.1‰ acetic acid (v/v) at a flow rate of 0.8mL/min. The precursor and product ions of analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in negative mode using multiple-reaction monitoring (MRM). Full validation of the assay was carried out including linearity, precision, accuracy, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid and specific. Then the method was successfully applied to differentiate 18 batches of Herba Scutellariae barbatae samples and 3 batches of its confused plants from different sources.

Determination of sugammadex in human plasma, urine, and dialysate using a high-performance liquid chromatography/tandem mass spectrometry assay

Sugammadex (Bridion ®, Merck Sharp & Dohme Corp., Oss, The Netherlands) is a modified γ-cyclodextrin which has the ability to reverse the neuromuscular blockade induced by the steroidal neuromuscular blocking agents rocuronium and vecuronium. The objective of the current study is to describe the bioanalytical methods that have been developed and validated according to US Food and Drug Administration guidelines on bioanalytical method validation, and subsequently applied to determine total sugammadex (i.e., free sugammadex plus sugammadex bound to the neuromuscular blocking agent) in human heparinized plasma, urine and dialysate. Sugammadex was extracted from human plasma and urine using solid phase extraction with Isolute HAX 96-well extraction plates; no extraction was performed on dialysate samples. Samples from plasma, urine, and dialysate were analyzed on a Polaris ® C18-A PEEK (polyaryletheretherketone) analytical column (50 mm × 4.6 mm internal diameter, 5 μm) with a linear mobile phase gradient of 0.1% (v/v) formic acid in water:methanol from 70:30 to 20:80. The flow rate was 1 mL/min with a total run time for each injection of 6 min. Tandem mass spectrometric detection was conducted using multiple reaction monitoring under negative ion mode with a turbo ion-spray interface to quantify the concentration of sugammadex. Inter- and intra-assay precision and accuracy were within pre-defined acceptance limits. The presence of rocuronium did not interfere with the assay in plasma, urine or dialysate; similarly, vecuronium did not interfere with the plasma assay (not tested for interference in urine or dialysate). Sugammadex was found to be stable in plasma, urine and dialysate in the short-term at room temperature, in the long-term at −20 °C, and after several freeze/thaw cycles. The validated bioanalytical methods developed here have been successfully applied in a series of clinical studies for the determination of total sugammadex in plasma, urine and dialysate.

Naturally Occurring Human Urinary Peptides for Use in Diagnosis of Chronic Kidney Disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Quantitative analysis of nine coumarins in rat urine and bile after oral administration of Radix Glehniae extract by high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry

Coumarins are the primary bioactive ingredients in Radix Glehniae, named Beishashen in China, which possesses many pharmacological activities, including anticancer, anti-inflammation and antivirus activities. In the present study, we employed a sensitive and selective high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of nine coumarins in rat urine and bile: scopoletin (1), xanthotoxol (2), xanthotoxin (3), psoralen (4), isoimpinellin (5), bergapten (6), oxypeucedanin (7), imperatorin (8) and isoimperatorin (9). Pimpinellin (10) was used as the internal standard (IS). The urine and bile samples were pretreated by liquid-liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C₁₈ column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in positive mode using multiple-reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect and several stabilities were validated for coumarins in rat urine and bile samples. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of the excretion study of the nine coumarins in Radix Glehniae.

Stability‐indicating validation of acitretin and isoacitretin in human plasma by LC‐ESI‐MS/MS bioanalytical method and its application to pharmacokinetic analysis

LC- ESI- MS/MS simultaneous bioanalytical method was developed to determine acitretin and its metabolite isoacitretin in human plasma using acitretin-d3 used as the internal standard for both analytes. The compounds were extracted using protein precipitation coupled with liquid-liquid extraction with flash freezing technique. Negative mass transitions (m/z) of acitretin, isoacitretin and acitretin-d3 were detected in multiple reactions monitoring (MRM) mode at 325.4 → 266.3, 325.2 → 266.1 and 328.3 → 266.3, respectively, with a turbo ion spray interface. The chromatographic separation was achieved on an Ascentis-RP amide column (4.6 × 150 mm, 5 µm) with mobile phase delivered in isocratic mode. The method was validated over a concentration range of 1.025-753.217 ng/mL for acitretin and 0.394-289.234 ng/mL for isoacitretin with a limit of quantification of 1.025 and 0.394 ng/mL. The intra-day and inter-day precisions were below 8.1% for acitretin and below 13.8% for isoacitretin, while accuracy was within ±7.0 and ±10.6% respectively. For the first time, the best possible conditions for plasma stability of acitretin and isoacitretin are presented and discussed with application to clinical samples.

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry

A simple offline LC–MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm × 50 mm, 3 μm) using a mobile phase of ACN/H 2O (80/20, v/v) containing 10 mM NH 4Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/ z 408 → 235 for sitagliptin and m/ z 412 → 239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 μL of plasma is processed. The linear calibration range is 1–1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n = 6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.

Simultaneous quantification of 19 diterpenoids in Isodon amethystoides by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method was developed to characterize and quantify 19 diterpenoid compounds in Isodon amethystoides simultaneously. By employing a Diamonsil C 18 column, 19 constituents were separated within 15 min using a gradient elution consisted of methanol containing 0.1% formic acid and 0.1% aqueous formic acid. The precursor and product ions of the analytes were monitored on a hybrid quadrupole linear ion trap mass spectrometer equipped with a turbo ion spray interface in positive and negative mode in a single run and quantified by a multiple-reaction monitor (MRM). All standard calibration curves showed good linearity ( r 2 > 0.99) within the test ranges. The precision was evaluated by intra- and inter-day tests, which revealed relative standard deviation (RSD) values within the ranges of 1.06–3.25% and 1.56–3.84%. The recovery studies for the quantified compounds were between 95.82 and 108.3% with RSD values less than 1.86%. The results indicated that the method is simple, rapid, specific and reliable. This method was successfully applied for identification and quantification of 19 diterpenoids in 11 batches of I. amethystoides. The results showed that the contents of diterpenoids in I. amethystoides from different sources were widely varied.

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/ z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.

LC-MS-ESI for the Determination of Loratadine and Descarboethoxyloratadine in Human Plasma

A rapid, sensitive, and accurate liquid chromatography-tandem mass spectrometry assay for simultaneous determination of loratadine (L) and its active metabolite, descarboethoxyloratadine (DCL), in human plasma is developed using desipramine as internal standard (IS). The analytes and IS are separated on a Betabasic cyano (100 mm x 2.1 mm, 5 microm) column and detected by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime is 3.0 min with retention time for L, DCL, and IS at 0.82, 1.58, and 1.97 min, respectively. The method is validated over a dynamic linear range of 0.05-15.00 ng/mL for both L and DCL with a correlation coefficient of r(2) 0.9984 and 0.9979, respectively. The intra-batch and inter-batch precision (%CV) across five levels (LLOQ, LQC, MQC, HQC, and ULOQ) is less than 9%. The method is successfully applied to a bioequivalence study of 10 mg loratadine tablet formulation in 28 healthy Indian male subjects under fasted condition.

Simultaneous determination of propranolol and 4-hydroxy propranolol in human plasma by solid phase extraction and liquid chromatography/electrospray tandem mass spectrometry

A very sensitive, reliable, reproducible and highly selective assay for the simultaneous determination of free and total (conjugated and unconjugated) propranolol and its equipotent hydroxyl metabolite, 4-hydroxy propranolol, in human plasma was developed and validated. The analytes were simultaneously extracted from 0.300 mL of human plasma using solid phase extraction and detected in positive ion mode by tandem mass spectrometry with a turbo ionspray interface. Deuterium-labeled propranolol and 4-hydroxy propranolol, propranolol-d7 and 4-hydroxy propranolol-d7, were used as internal standards. The method has a lower limit of quantitation (LOQ) of 0.20 ng/mL for both analytes with the limits of detection (LOD) 50 and 100 pg/mL for propranolol and 4-hydroxy propranolol, respectively, based on a signal-to-noise ratio of 5. The assay was linear over a range 0.20–135.00 ng/mL for free propranolol and 0.20–25.00 ng/mL for free 4-hydroxy propranolol and linear over range 1.00–500.00 ng/mL for total propranolol and 1.00–360.00 ng/mL for total 4-hydroxy propranolol, with coefficient of determination greater than 0.99 for both analytes. The extraction recoveries were >96 and >64% on an average for propranolol and 4-hydroxy propranolol, respectively. The analytes were found stable in human plasma through five freeze (−15 °C)–thaw (room temperature) cycles and under storage on bench-top for at least 6.5 h, and also in mobile phase at 10 °C for at least 48 h. The method has shown tremendous reproducibility, with intra- and inter-day precision <11.3% (RSD), and intra- and inter-day accuracy <11% of nominal values, for both analytes, and has proved to be highly reliable for the analysis of clinical samples.

The Origins of Ion Spray Liquid Chromatography–Tandem Mass Spectrometry

Featured Article: Bruins AP, Covey TR, Henion JD. Ion spray interface for combined liquid chromatography/atmospheric pressure ionization mass spectrometry. Anal Chem 1987;59:2642–6.1 The 1987 paper highlighted here evolved from the ongoing desire to couple HPLC to mass spectrometry. HPLC-ultraviolet techniques were readily accepted and widely used, but the facile and practical coupling of this very capable separation science technique to electrospray ionization mass spectrometry had not yet been demonstrated. During the late 1970s only a handful of us were experimenting with coupling HPLC to mass spectrometry to provide LC-MS capabilities. In 1979 Hewlett-Packard developed an early approach called direct liquid introduction (DLI)2 to provide one of first commercialized LC-MS interfaces. At the same time Finnigan, Inc. commercialized its “moving belt” LC-MS interface, which could produce electron ionization (EI) mass spectra via LC-MS. Neither of these commercial LC-MS techniques was easy to use or generally accepted as a useful analytical tool. During the early 1980s a few research groups continued to explore alternative approaches to create gas-phase ions from intractable compounds for mass spectrometry (MS) detection …

Simultaneous screening and quantification of 25 opioid drugs in post-mortem blood and urine by liquid chromatography–tandem mass spectrometry

A method for simultaneous screening and quantification was developed for the fentanyls alfentanil, fentanyl, p-fluorofentanyl, cis-3-methylfentanyl, trans-3-methylfentanyl, alpha-methylfentanyl, norfentanyl, remifentanil, sufentanil, and the other opioid drugs 6-acetylmorphine, buprenorphine, codeine, dextropropoxyphene, ethylmorphine, heroin, methadone, morphine, naloxone, naltrexone, norbuprenorphine, normethadone, oxycodone, pentazocine, pethidine, and tramadol in post-mortem blood and urine samples by LC–MS/MS. Samples were extracted with butyl acetate at pH 7. The drugs were separated by LC on a Genesis C 18 reversed-phase column, with a gradient consisting of acetonitrile and ammonium acetate at pH 3.2. The mass spectrometric analysis was performed with a quadrupole-linear ion-trap mass spectrometer equipped with a turbo ion spray interface in positive mode using multiple reaction monitoring (MRM). Quantification was performed based on five isotope-labelled internal standards. Validation included assessment of linearity, limit of quantification, inaccuracy, precision, and matrix effects. The limits of quantification were adequate for screening and quantification of opioid drugs at low therapeutic or abuse concentration levels, with inaccuracy less than 23% and precision better than 24% both in blood and urine samples. When this method was applied to autopsy cases, its results were in agreement with those of reference methods.

Determination of Low-Level Acrylamide in Drinking Water by Liquid Chromatography/Tandem Mass Spectrometry

A simple and sensitive liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method has been developed and validated to confirm and quantify acrylamide monomer (AA) in drinking water using [13C3] acrylamide as internal standard (IS). After a preconcentration by solid-phase extraction with spherical activated carbon, analytes were chromatographed on IonPac ICE-AS1 column (9 x 250 mm) under isocratic conditions using acetonitrile-water-0.1 M formic acid (43 + 52 + 5, v/v/v) as the mobile phase. Analysis was achieved using a triple-quadrupole mass analyzer equipped with a turbo ion spray interface. For confirmation and quantification of the analytes, MS data acquisition was performed in the multireaction monitoring mode, selecting 2 precursor ion to product ion transitions for both AA and IS. The method was validated for linearity, sensitivity, accuracy, precision, extraction efficiency, and matrix effect. Linearity in tap water was observed over the concentration range 0.1-2.0 microg/L. Limits of detection and quantification were 0.02 and 0.1 microg/L, respectively. Interday and intraday assays were performed across 3 validation levels (0.1, 0.5, and 1.5 microg/L). Accuracy (as mean recovery) ranged from 89.3 to 96.2% with relative standard deviation <7.98%. Performance characteristics of this LC/MS/MS method make it suitable for regulatory confirmatory analysis of AA in drinking water in compliance with European Union and U.S. Environmental Protection Agency standards.

Highly sensitive and rapid LC–ESI-MS/MS method for the simultaneous quantification of uroselective α 1-blocker, alfuzosin and an antimuscarinic agent, solifenacin in human plasma

An accurate, selective and sensitive bioanalytical method has been developed and validated for the simultaneous quantification of alfuzosin and solifenacin in human plasma using propranolol as internal standard (IS). The analytes and IS were extracted in methyl tert-butyl ether, separated on Hypurity C8 column and detected by tandem mass spectrometry with a turbo ion spray interface. The method had a chromatographic run time of 3.0 min and linear calibration curves over the concentration range of 0.25–25 ng/mL for alfuzosin and 0.6–60 ng/mL for solifenacin. The intra- and inter-day accuracy and precision (%CV) evaluated at four quality control levels were within 88.2–106.4% and 0.9–7.7% respectively. The absolute recovery from spiked plasma samples was 71.8% for alfuzosin and 93.1% for solifenacin. Stability of alfuzosin and solifenacin was assessed under different storage conditions. The validated method was successfully employed for bioavailability study after oral administration of 10 mg of alfuzosin hydrochloride and 5 mg of solifenacin succinate tablet formulations in eight healthy volunteers under fed condition.

High‐throughput LC‐MS/MS assay for 6‐methoxy‐2‐naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study

A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition.

Validated hydrophilic interaction LC–MS/MS method for simultaneous quantification of dacarbazine and 5-amino-4-imidazole-carboxamide in human plasma

A hydrophilic interaction high performance liquid chromatography–tandem mass spectrometric method has been developed and validated for simultaneous quantification of dacarbazine (DTIC) and its terminal metabolite, 5-amino-4-imidazole-carboxamide (AIC) in human plasma. The plasma samples are first extracted by a C8 + SCX mixed-mode 96-well plate to extend the extraction stability of DTIC and AIC. The extracted residues are further cleaned by a primary and secondary amine (PSA) adsorbent for minimization of matrix effect. Analyses are done on an Amide-80 HPLC column coupled to a tandem mass spectrometer fitted with an atmospheric pressure turbo ion spray ionization interface in the positive-ion mode. Both DTIC and AIC have reproducible retention times on the Amide-80 HPLC column. This type of column not only has an excellent column life (over 4000 injections), but also has zero carryover effect. The injection volume should be limited at 10 μL or less to avoid the peak splitting. The validated concentration ranges are from 0.5 to 500 ng/mL for DTIC and from 2.0 to 500 ng/mL for AIC. The validated method has been successfully applied to determine the pharmacokinetic profiles for human patients receiving DTIC infusions.

Tetrodotoxin poisoning evidenced by solid-phase extraction combining with liquid chromatography–tandem mass spectrometry

The toxicity and toxin component of gastropod Niotha clathrata implicated to a food paralytic poisoning incident in Kaohsiung, Taiwan in November 2006 were studied. The highest scores of average toxicity in the digestive gland and other portions from collected gastropods were 62 ± 24 (mean ± S.D.) and 32 ± 16 μg/g according to tetrodotoxin (TTX) bioassay, respectively. The toxin from these gastropods was large amount and easily identified as tetrodotoxin by traditional method of HPLC-FLD. The toxin of patient's blood serum was trace amount and analyzed by a new developed method LC–MS/MS. LC–MS/MS was contracted by the LC system interfaced with the MS/MS system with a turbo ion spray interface. Positive ion detection and multiple reaction monitoring mode were used for TTX of patient serum. It was found that linearity in serum was observed within concentration ranged of 1–100 ng/ml and limit of detection was 0.1 ng/ml. The LOQ was reproducible at 1 ng/ml in serum. The blood serum showed to contain TTX of 3.30 ± 0.08 ng/ml. It indicated that LC–MS/MS was more lower detectable and believable method for TTX determination than LC–MS reported previously. Furthermore, the causative agent of gastropod food poisoning was identified as TTX.

HPLC–ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm × 4.6 mm i.d., 3.5 μm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5–150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1–150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5–109.1% and 3.0–14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were ≥90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.

Simultaneous determination of simvastatin and simvastatin acid in human plasma by LC‐MS/MS without polarity switch: Application to a bioequivalence study

A simple, specific and sensitive LC-MS/MS assay for simultaneous determination of simvastatin (SV) and its active beta-hydroxy acid metabolite, simvastatin acid (SVA) in human plasma was developed using a statin analog as internal standard (IS). The method was validated over a dynamic linear range of 0.20-100.00 ng/mL for SV and 0.10-50.00 ng/mL for SVA with correlation coefficient r > or = 0.9987 and 0.9989, respectively. The analytes and IS were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction using methyl tert-butyl ether and separated through an Aquasil C18 column (100 mm x 2.1 mm, 5 microm). Detection of analytes and IS was done by MS/MS with a turbo ion spray interface operating in positive ion and selective reaction monitoring acquisition mode. The total chromatographic run time was 3.0 min. Flash freezing of the aqueous phase was an added advantage during liquid-liquid extraction, which considerably reduced time and labour. The method was extensively validated for its accuracy, precision, recovery, stability studies and matrix effect. The method was successfully used for bioequivalence study of 40 mg SV tablet formulation in 12 human subjects under fasting condition.