Ultra-sensitive determination of Formoterol in human serum by high performance liquid chromatography and electrospray tandem mass spectrometry

An analytical method was developed and validated to determine Formoterol in human serum in the range from 0.40 to 100.24 pg/mL by high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) due to the lack of efficient methods to determine very low levels of Formoterol in serum and plasma. Serum was diluted by water and mixed with the internal standard (d6-Formoterol). Formoterol and internal standard were extracted using a cation-exchange solid phase column (SCX-3). After eliminating endogenous serum constituents through washing steps with water and methanol, elution took place using methanol/ammonia. After evaporation of the elution liquid the residue was redissolved and analyzed by HPLC-MS/MS with electrospray ionisation (ESI) in positive mode. A gradient between 10 mM ammonium formate and acetonitrile was used. The inter-batch precision of the calibration standards ranged from 1.55% to 9.01%. The inter-batch accuracy of the calibration standards ranged from 93.37% to 107.30%. The lower limit of quantitation (LLOQ, 0.40 pg/mL) had a precision of 19.67% and an accuracy of 96.78%. Comparable results were obtained for quality control samples. Stability in human serum was given over three freeze/thaw cycles and 2h at room temperature. Formoterol in human serum was stable for at least 6 months below -20 degrees C. This method has been used widely for quantifying Formoterol after inhalation of 9-36 microg of the drug by volunteers. A cross validation with human plasma versus serum was performed after this method was successfully validated in human serum.     

The development of a C1q-solid-phase immunoenzyme method for determining circulating immune complexes]

The method of quantitative enzyme immunoassay (EIA) for the determination of circulating immune complexes (CIC) was developed on the basis of solid-phase human C1q. The calibration curve was plotted with the use of aggregated human gamma-globulin (AHGG), the optimum range of concentration being 15-500 microg/ml. In the process of approbation on clinical material the method revealed an elevated level of CIC in the sera of patients in comparison with their level in the sera of healthy donors. Out of 40 studied serum samples from patients with Yersinia infection, in 3 serum samples the levels of CIC was 26, 65 and 94 microg of AHGG equivalents per ml. In 4 out of 46 studied serum samples obtained from patients with diagnosed Yersinia arthritis the level of CIC was 12, 27, 46 and 186 microg of AHGG per ml, and in serum samples from healthy donors this level was 8.6 microg/ml [corrected].     

Quantitative determination of the diastereoisomers of hexabromocyclododecane in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A sensitive, simple and feasible method has been developed and validated for the simultaneous determination of three diastereoisomers of hexabromocyclododecane (HBCD) in human plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). The simple pretreatment generally involved protein precipitation with methanol (MeOH). The separation was performed with a C18 reverse phase column. The mobile phases were 5mM ammonium acetate (NH(4)AC) in water and acetonitrile (ACN). The mass spectrometer was operated using negative electrospray ionization (ESI) source and the data acquisition was carried out with multiple reaction monitoring (MRM) mode. The analyte quantifications were performed by external standard method with matrix-matched calibration curves. The method was partially validated with the evaluations of accuracy, precision, linearity, limit of quantification (LOQ), limit of detection (LOD), recovery, matrix effect and carryover effect. With the present method, the intra-batch accuracies were 94.7-104.3%, 91.9-109.3% and 89.8-105.0% for α-, β- and γ-HBCD, respectively. And the inter-batch accuracies were ranged from 94.2% to 109.7%. Both intra-batch and inter-batch precisions (relative standard deviation, RSD, %) of the analytes were no more than 11.2%. The recoveries were from 79.0% to 108.9% and the LOQ was 10pg/mL for each diastereoisomer. The linear range was 10-10,000pg/mL with the linear correlation coefficient R(2)>0.996. No significant matrix effect and carryover effect of the analytes were observed in this study. This method is in possession of sufficient resolution, high sensitivity as well as selectivity and convenient to be applied to the trace determination of HBCDs in human plasma.     

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 x 4.6 mm i.d., 3.5 microm 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 > or = 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.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and alprazolam in human plasma by liquid chromatography-electrospray ionization mass spectrometry

A fast liquid chromatographic assay with mass spectrometric detection (LC/MS) has been developed and validated for the simultaneous determination of methadone (MT), its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and alprazolam, in human plasma. The extraction procedure was performed with automatic solid phase extraction, and the compounds were separated with a Sunfire column using a gradient mode. Deuterated analogues for all of the analytes of interest were used for quantitation. Limits of detection (LOD) were established between 0.5 and 1 ng/ml. Linearity was obtained over a range of 2-2,000 ng/ml with an average correlation coefficient (R(2)) of >0.99. Intra- and inter-batch coefficients of variation and relative mean errors were less than 10% for all analytes and concentrations. The recoveries were higher than 50.0% in all cases. The method proved to be suitable for evaluation of plasma obtained from patients enrolled in a MT-maintenance program who are frequently treated with alprazolam as a sedative.     

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.     

In vitro metabolism of beclomethasone dipropionate, budesonide, ciclesonide, and fluticasone propionate in human lung precision-cut tissue slices

Background

The therapeutic effect of inhaled corticosteroids (ICS) may be affected by the metabolism of the drug in the target organ. We investigated the in vitro metabolism of beclomethasone dipropionate (BDP), budesonide (BUD), ciclesonide (CIC), and fluticasone propionate (FP) in human lung precision-cut tissue slices. CIC, a new generation ICS, is hydrolyzed by esterases in the upper and lower airways to its pharmacologically active metabolite desisobutyryl-ciclesonide (des-CIC).

Methods

Lung tissue slices were incubated with BDP, BUD, CIC, and FP (initial target concentration of 25 μM) for 2, 6, and 24 h. Cellular viability was assessed using adenosine 5'-triphosphate content and protein synthesis in lung slices. Metabolites and remaining parent compounds in the tissue samples were analyzed by HPLC with UV detection.

Results

BDP was hydrolyzed to the pharmacologically active metabolite beclomethasone-17-monopropionate (BMP) and, predominantly, to inactive beclomethasone (BOH). CIC was hydrolyzed initially to des-CIC with a slower rate compared to BDP. A distinctly smaller amount (approximately 10-fold less) of fatty acid esters were formed by BMP (and/or BOH) than by BUD or des-CIC. The highest relative amounts of fatty acid esters were detected for BUD. For FP, no metabolites were detected at any time point. The amount of drug-related material in lung tissue (based on initial concentrations) at 24 h was highest for CIC, followed by BUD and FP; the smallest amount was detected for BDP.

Conclusion

The in vitro metabolic pathways of the tested ICS in human lung tissue were differing. While FP was metabolically stable, the majority of BDP was converted to inactive polar metabolites. The formation of fatty acid conjugates was confirmed for BMP (and/or BOH), BUD, and des-CIC.     

Determination of mycophenolic acid glucuronide in microsomal incubations using high performance liquid chromatography-tandem mass spectrometry

A sensitive and specific HPLC-MS/MS method was developed for the analysis of mycophenolic acid glucuronide (MPAG) in incubations with human liver microsomes. Incubation samples were processed by protein precipitation with acetonitrile. MPAG and the internal standard phenolphthalein glucuronide were chromatographed on a C18 Synergi Fusion-RP column (100 mm x 2 mm, 4 microm) using gradient elution with a mixture of 1mM acetic acid in deionized water and 1mM acetic acid in acetonitrile at a flow rate of 0.22 mL/min. The mass spectrometer was operated with negative electrospray ionization and analysis was carried out in the single reaction monitoring (SRM) mode using the mass transitions of m/z 495-->319 and m/z 493-->175 for MPAG and phenolphthalein glucuronide, respectively. The MPAG calibration curve was linear over the concentration range of 1.0-20 microM. The within-day and between-day relative standard deviations ranged from 1.1 to 7.9% and accuracy was within 8%. The simple and reproducible method is suitable for measuring mycophenolic acid glucuronidation in microsomal incubations.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

High-performance liquid chromatography-electrospray mass spectrometry for the simultaneous determination of multiple active components in Sheng-Mai San, a prescription of traditional Chinese medicine

An advanced and reliable HPLC-MS method was developed for the simultaneous quantification of eight active components (ginsenosides Rf, Rg(2), Rg(3), Rh(1) and Rh(2), gomisin A, methylophioponanone B and schizandrin) in Sheng-Mai San, a traditional Chinese medicine. The elution of multiple components was performed using a C(18) column with stepwise gradient elution. The detection of individual analytes was monitored by electrospray MS scanning from 300 to 1000 m/z in the positive ion mode, with the limits of detection of these components ranging from 0.06 to 1 microg/mL at a signal-to-noise ratio of > or =5. The intra- and inter-day accuracies ranged from 95.1 to 104.4%, and the overall precision was less than 9.3%. The recoveries of the analytes were > or =96.6%. The method was validated and found suitable for the determination of active components present in Sheng-Mai San preparation.     

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.     

High throughput and sensitive determination of trazodone and its primary metabolite, m-chlorophenylpiperazine, in human plasma by liquid chromatography-tandem mass spectrometry

A precise, sensitive and high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of trazodone (TRZ) and its primary metabolite, m-chlorophenylpiperazine (mCPP), in human plasma was developed and validated. The analytes and the internal standard-nefazodone were extracted from 500 microL aliquots of human plasma via liquid-liquid extraction in n-hexane. Chromatographic separation was achieved in a run time of 2.5 min on a Betabasic cyano column (100 mm x 2.1 mm, 5 microm) under isocratic conditions. Detection of analytes and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for TRZ, mCPP and IS were m/z 372.2-->176.2, 197.2-->118.1 and 470.5-->274.6 respectively. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, stability study and dilution integrity. A linear dynamic range of 10.0-3000.0 ng/mL for TRZ and 0.2-60.0 ng/mL for mCPP was evaluated with mean correlation coefficient (r) of 0.9986 and 0.9990 respectively. The intra-batch and inter-batch precision (%CV) across five validation runs (LLOQ, lower limit of quantitation; LQC, low quality control; MQC, middle quality control; HQC, high quality control and ULOQ, upper limit of quantitation) was < or =8.4% for both the analytes. The method was successfully applied to a bioequivalence study of 100mg trazodone tablet formulation in 36 healthy Indian male subjects under fasting and fed conditions.     

Identification and determination of major flavonoids in rat serum by HPLC-UV and HPLC-MS methods following oral administration of Dalbergiaodorifera extract

The major flavonoids in rat serum after oral administration of Dalbergia odorifera extract were analyzed qualitatively and quantitatively by high performance liquid chromatography (HPLC) and its coupling to mass spectrometry (HPLC-MS). Utilizing HPLC-MS technique, 18 flavonoids including five isoflavones, four isoflavanones, four neoflavones, two flavanones, two chalcones, one isoflavanonol were identified in free form in serum sample based on comparison with the authentic standards. Furthermore, the amounts of the four prominent flavonoids, (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone, vestitone, formononetin and sativanone were determined in serum by HPLC-UV with internal standard method. The method was validated and utilized in pharmacokinetic studies of these four analytes. This is the first report on identification and determination of the major flavonoids in rat serum after oral administration of D. odorifera extract and the results provided a firm basis for clarifying the pharmacological effect of D. odorifera and evaluating the clinical applications of this medicinal herb.     

Simultaneous determination of ten antiarrhythic drugs and a metabolite in human plasma by liquid chromatography--tandem mass spectrometry

A simple, accurate and selective LC-MS/MS method was developed and validated for simultaneous quantification of ten antiarrhythic drugs (diltiazem, amiodarone, mexiletine, propranolol, sotalol, verapamil, bisoprolol, metoprolol, atenolol, carvedilol) and a metabolite (norverapamil) in human plasma. Plasma samples were simply pretreated with acetonitrile for deproteinization. Chromatographic separation was performed on a Capcell C(18) column (50mmx2.0mm, 5microm) using a gradient mixture of acetonitrile and water (both containing 0.02% formic acid) as a mobile phase at flow rate of 0.3ml/min. The analytes were protonated in the positive electrospray ionization (ESI) interface and detected in multiple reaction monitoring (MRM) mode. Calibration curves were linear over wide ranges from sub- to over-therapeutic concentration in plasma for all analytes. Intra- and inter-batch precision of analysis was <12.0%, accuracy ranged from 90% to 110%, average recovery from 85.0% to 99.7%. The validated method was successfully applied to therapeutic drug monitoring (TDM) of antiarrhythic drugs in routine clinical practice.     

Determination of talinolol in human plasma by high performance liquid chromatography-electrospray ionization mass spectrometry: application to pharmacokinetic study

A rapid and sensitive method for determination and screening in human plasma of talinolol is described using propranolol as the internal standard. The analytes in plasma were extracted by liquid-liquid extraction using methyl t-butyl ether. After removed and dried the upper organic phase, the extracts were reconstituted with a fixed volume of buffer of ammonium acetate and acetonitrile (60:40, v/v). The extracts were analyzed by a HPLC coupled to electrospray ionization mass spectrometry (HPLC-MS/ESI). The HPLC separation of the analytes was performed on a Phenomenex C18 (250 mmx4.6 mm, 5 microm, USA) column, with a flow rate of 0.85 mL/min. The complete elution was obtained within 5.5 min. The calibration curve was linear in the 1.0-400.0 ng/mL range for talinolol, with a coefficient of determination of 0.9996. The average extraction recovery was above 83%. The methodology recovery was between 101% and 102%. The limit of detection (LOD) was 0.3 ng/mL for talinolol. The intraday and inter-day coefficients of variation were less than 6%. This HPLC-MS/ESI procedure was used to assess the pharmacokinetics of talinolol. A single oral 50 mg dose of talinolol tablet was administered to 12 healthy Chinese volunteers, the main pharmacokinetic data are as follows: Cmax was 147.8+/-63.8 ng/mL; tmax was 2.0+/-0.7 h; t1/2 was 12.0+/-2.6 h. The method is accurate, sensitive and simple for the pharmacokinetic study of talinolol.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.     

Determination of antifilarial compound UMF-078 and its metabolites in plasma by high-performance liquid chromatography

UMF-078, methyl (±)-[5-(α-amino-4-fluorobenzyl)benzimidazol-2-yl]carbamate, is a new antifilarial compound being developed by the World Health Organization. In the present study, a HPLC method for the simultaneous estimation of UMF-078 and its metabolites (flubendazole, decarbamoylated flubendazole, UMF060 and decarbamoylated UMF-060) in plasma was developed, validated and applied to pharmacokinetic studies. Linearity was observed between 20 and 1000 ng/ml for decarbamoylated UMF-060 and between 10 and 500 ng/ml for other analytes. Recoveries were consistent over the concentration ranges studied for all the analytes. Variations in intra- and inter-batch accuracy and precision were within acceptable limits of ±20% at the lowest limit of quantitation, whereas at higher concentrations it was ±15%. The analytes showed stability up to two freeze–thaw cycles in plasma. No degradation was observed for any of the analytes even after 72 h of storing the dry plasma extracts at −30°C. The assay method was employed to study the pharmacokinetics of hydrochloride salt of UMF-078 in rats. The parent compound and its metabolites viz: decarbamoylated UMF-060, UMF-060 and flubendazole were quantitated in serum and the compounds could be monitored up to 168 h post-dose.     

Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry

Clozapine (CLZ), olanzapine (OLZ), risperidone (RIP) and quetiapine (QTP) have been widely used in the treatment of schizophrenia. However, no study (or little study) has been conducted to determine the four drugs simultaneously by the use of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI). OBJECTIVE: To develop a sensitive method for simultaneous determination of CLZ, OLZ, RIP and QTP in human plasma by HPLC-MS/ESI. METHODS: The analytes were extracted twice by ether after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C(18) ( [Formula: see text] mm, 3 microm, Germany) column, using water (formic acid: 2.70 mmol/l, ammonium acetate: 10 mmol/l)-acetonitrile (53:47) as mobile phase, with a flow-rate of 0.16 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. RESULTS: The calibration curves were linear in the ranges of 20-1000 ng/ml for CLZ and QTP, 1-50 ng/ml for OLZ and RIP, respectively. The average extraction recoveries for all the four analysts were at least above 80%. The methodology recoveries were higher than 91% for the analysts. The intra- and inter-day R.S.D. were less than 15%. CONCLUSION: The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) and for the study of the pharmacokinetics of the four drugs.     

Method for the simultaneous determination of losartan and its major metabolite, EXP-3174, in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometric method was developed for the simultaneous determination of losartan and its major active metabolite, EXP-3174, in human plasma. The two analytes and the internal standard (DuP-167) were extracted from plasma under acidic conditions by using solid-phase extraction cartridges containing a sorbent of copolymer, poly(divinylbenzene-co-N-vinylpyrrolidone). The analytes were separated by LC equipped with a reversed-phase C₁₈ column, and introduced into the mass spectrometer via the electrospray ion source with pneumatically-assisted nebulization. For LC–MS–MS samples, an isocratic mobile phase consisting of [0.1% triethylamine–0.1% acetic acid (pH 7.1)]–acetonitorile (65:35, v/v) was used, and the assay was monitored for the negative fragment ions of the analytes. The method demonstrated linearity from 1 to 1000 ng/ml for both losartan and EXP-3174. The limit of quantification for both compounds in plasma was 1 ng/ml. This assay method may be useful for the measurement of levels of the two compounds in clinical studies of losartan.