Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

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/H₂O (80/20, v/v) containing 10 mM NH₄Ac (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.     

Chiral analysis of ammuxetine enantiomers in dog plasma using online SPE/liquid chromatography with tandem mass spectrometric detection after precolumn chiral derivatization

Ammuxetine (AMT), a novel chiral antidepressant candidate compound, exhibits better antidepression effects than duloxetine in different animal models. In this article, a chiral derivatization method, combined with online solid phase extraction (online SPE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), was developed for the chiral separation of AMT enantiomers after administration of racemic AMT to dogs. The derivatization reaction employed 2,3,4,6‐tetra‐O‐acetyl‐b‐glucopyr‐anosyl isothiocyanate (GITC) as a precolumn chiral derivatization reagent. A SPE column Retain PEP Javelin (10 × 2.1 mm) was used to remove proteins and other impurities in plasma samples. The enantiomeric derivatives were separated on a ZORBAX SB‐C₁₈ column (50 × 2.1 mm × 3.5 μm) with an isocratic elution procedure. The selected multiple reaction monitoring mode of the positive ion was performed and the parent to the product transitions m/z 681.0/543.1 and m/z 687.4/543.1 were used to measure the derivatives of AMT and duloxetine (internal standard) with electrospray ionization. The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The method was applied to a pharmacokinetics study of AMT racemate in dogs. The results suggested that the pharmacokinetic of AMT enantiomers might be stereoselective in dogs.     

Determination of norgestimate and its metabolites in human serum using high-performance liquid chromatography with tandem mass spectrometric detection

A rapid and reliable analytical method is described for the simultaneous determination of a synthetic progestin norgestimate (NGM), and its metabolites, 17-deacetylnorgestimate (17-DA-NGM), 3-ketonorgestimate (3-keto-NGM) and norgestrel (NGL) in human serum using reversed phase high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The assay was linear over the concentration ranges of 0.1–5.0 ng/ml for 17-DA-NGM and NGL and 0.5–5.0 ng/ml for NGM and 3-keto-NGM. The inter-assay reproducibility was consistently less than 10%. The overall recovery of the analytes ranged from 72 to 92%. Serum profiles following oral administration of norgestimate to female volunteers are presented.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of talinolol in human plasma using automated on-line solid phase extraction combined with atmospheric pressure chemical ionization tandem mass spectrometry

A specific LC-MS/MS assay was developed for the automated determination of talinolol in human plasma, using on-line solid phase extraction system (prospekt 2) combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. The method involved simple precipitation of plasma proteins with perchloric acid (contained propranolol) as the internal standard (IS) and injection of the supernatant onto a C8 End Capped (10 mmx2 mm) cartridge without any evaporation step. Using the back-flush mode, the analytes were transferred onto an analytical column (XTerra C18, 50 mmx4.6 mm) for chromatographic separation and mass spectrometry detection. One of the particularities of the assay is that the SPE cartridge is used as a column switching device and not as an SPE cartridge. Therefore, the same SPE cartridge could be used more than 28 times, significantly reducing the analysis cost. APCI ionization was selected to overcome any potential matrix suppression effects because the analyte and IS co-eluted. The mean precision and accuracy in the concentration range 2.5-200 ng/mL was found to be 103% and 7.4%, respectively. The data was assessed from QC samples during the validation phase of the assay. The lower limit of quantification was 2.5 ng/mL, using a 250 microL plasma aliquot. The LC-MS/MS method provided the requisite selectivity, sensitivity, robustness accuracy and precision to assess pharmacokinetics of the compound in several hundred human plasma samples.     

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.     

Determination of deracoxib in feline plasma samples using high performance liquid chromatography

A new HPLC procedure for the determination of deracoxib, a selective cyclooxygenase-2 inhibitor, has been developed and validated. Following a liquid-liquid extraction using isopropyl alcohol and chloroform, samples were separated by isocratic reversed-phase HPLC on an Atlantis C18 column and quantified using UV detection at 252 nm. The mobile phase was a mixture of 10 mM potassium phosphate (pH 4.5) and acetonitrile, with a flow-rate of 1.0 ml/min. The procedure produced a linear curve over the concentration range 10-1500 ng/ml. The development of the assay allowed the determination of pharmacokinetic parameters after oral administration of deracoxib in cats and would be suitable for other pharmacokinetic studies.     

Validation and implementation of a method for determination of bryostatin 1 in human plasma by using liquid chromatography/tandem mass spectrometry

A new sensitive and specific method using liquid chromatography/tandem mass spectrometry for determination of bryostatin 1 was developed and validated. Sample pretreatment involved a double liquid-liquid extraction step with a mixture of acetonitrile/n-butyl chloride (1/4, v/v). Separation of the compound of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C18 (50 x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 mL/min for 13 min. The analytes of interest were monitored by tandem mass spectrometry with electrospray positive ionization. The linear calibration curves were generated over the range of 50-2000 pg/mL with values for the coefficient of determination of >0.99. The values for both within-day and between-day precision and accuracy were <15%. This method was used to characterize the plasma pharmacokinetics of bryostatin 1 at doses of 20 microg/m2) to optimize treatment with this agent.     

Determination of PF-04928473 in human plasma using liquid chromatography with tandem mass spectrometry

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) analytical method was developed for quantification of Hsp90 inhibitor PF-04928473 in human plasma, following administration of its prodrug, PF-04929113. Sample processing involved protein precipitation by addition of 0.4 mL of methanol containing internal standard (PF-04972487) to 50 μL volume of plasma sample. Chromatographic separation of PF-04928473 and PF-04972487 was achieved on a Phenomenex^® Luna C18(2) (2.0mm × 50 mm, 5 μm) column using a gradient elution method with mobile phase solvents: methanol containing 0.1% formic acid and 0.1% formic acid at a flow rate of 0.25 mL/min. Detection was performed in electrospray positive ionization mode, monitoring the ion transitions from m/z 465.1 → 350.1 (PF-04928473) and m/z 447.0 → 329.1 (PF-04972487). The retention times for PF-04928473 and PF-04972487 were 1.86 and 2.85 min, respectively. Calibration curves were generated in the range of 2–2000 ng/mL. The accuracy and precision ranged from 94.1 to 99.0% and 86.7 to 97.6%, respectively, which were calculated using quality control samples of three different concentrations analyzed in quintuplicate on four different days.     

Simultaneous determination of hydrocodone and hydromorphone in human plasma by liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous analysis of hydrocodone (HYC) and its metabolite hydromorphone (HYM) in human plasma. A robotic liquid handler and a 96-channel liquid handling workstation were used to aliquot samples, to add internal standard (I.S.), and to extract analytes of interest. A 96-well mixed-mode solid-phase cartridge plate was used to extract the analytes and I.S. The chromatographic separation was on a silica column (50 x 3 mm, 5-microm) with a mobile phase consisting of acetonitrile, water and trifluoroacetic acid (TFA) (92:8:0.01, v/v). The run time for each injection was 2.5 min with the retention times of approximately 2.1 and 2.2 min for HYC and HYM, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transition 300-->199 (m/z) for HYC, and 28-->185 (m/z) for HYM. The validated calibration curve range was 0.100-100 ng/ml, based on a plasma volume of 0.3 ml. The correlation coefficients were greater than or equal to 0.9996 for both HYC and HYM. The low limit of quantitation (LLOQ) was 0.100 ng/ml for both HYC and HYM with signal-to-noise ratio (S/N) of 50 and 10. respectively. The deuterated analytes, used as internal standards, were monitored at mass transitions 303-->199 (m/z) for HYC-d3 and 289-->185 (m/z) for HYM-d3. The inter-day (n= 17) precision of the quality control (QC) samples were < or = 3.5% RSD (relative standard deviation) for HYC and < or = 4.7% RSD for HYM, respectively. The inter-day accuracy of the QC samples were < or = 2.1% RE (relative error) for HYC and < or = 1.8% RE for HYM. The intra-day (n=6) precision and accuracy of the QC samples were < or = 2.6% RSD and < or = 3.0% RE for HYC, and < or = 4.7% RSD and < or = 2.4% RE for HYM. There was no significant deviation from the nominal values after a 5-fold dilution of high concentration QC samples by blank matrix. The QC samples were stable when kept at room temperature for 24-h or experienced three freeze-thaw cycles. The extraction recoveries were 86% for HYC and 78% for HYM. No detectable carryover was observed when a blank sample was injected immediately after a 2500 ng/ml sample that was 25-fold more concentrated than the upper limit of quantitation (ULOQ).     

Analysis of amphetamines and metabolites in urine with ultra performance liquid chromatography tandem mass spectrometry

A simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry method was developed and fully validated for the quantitative determination of seven amphetamines and metabolites in urine. The method was validated for selectivity, linearity, LOQ, LOD, imprecision, bias, analyte and processed sample stability, matrix effect, recovery, carryover and dilution integrity. A classic liquid–liquid extraction with ethyl acetate was used as sample preparation procedure. The compounds were separated on an Acquity UPLC HSS C₁₈ column in 6.8 min. The linear dynamic range was established from 25 to 500 ng/mL. The limit of quantification was fixed to the lowest calibrator level and the limit of detection ranged from 0.125 to 2.5 ng/mL. The method presented an excellent intra- and inter-assay imprecision and bias (<10.7%) at each measured concentration of two external quality controls (QC) and three “in house” QC. No matrix effects were observed and good recoveries (>70%) were obtained for all the compounds. No carryover was observed after the analysis of high concentrated samples (8000 ng/mL). The method was subsequently applied to authentic samples.     

Prenatal diagnosis for organic acid disorders using two mass spectrometric methods, gas chromatography mass spectrometry and tandem mass spectrometry

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.     

Detection of phencyclidine in human oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectrometric detection

An analytical procedure for the determination of phencyclidine in oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection, following initial screening with enzyme linked immunosorbent assay. The oral fluid samples were collected using the Quantisal device, and any drugs present were quantified using mixed mode solid-phase extraction followed by mass spectrometric detection in positive atmospheric pressure chemical ionization mode. For confirmation, two transitions were monitored and one ratio determined, which had to be within 20% of that of the known calibration standard. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 5ng/mL; the intra-day precision of the assay (n=5) was 3.04%; inter-day precision 3.35% (n=5) at a concentration of 10ng/mL. The accuracy was determined at four concentrations (5, 10, 20 and 40ng/mL) within the linear range of the assay. The percentage recovery of phencyclidine from the oral fluid collection pad was 81.7% (n=6). The methods were applied to both proficiency specimens and to samples obtained during research studies in the USA.     

Determination of glimepiride in human plasma using semi-microbore high performance liquid chromatography with column-switching

A fully automated semi-microbore high performance liquid chromatographic (HPLC) method with column-switching using UV detection was developed for the determination of glimepiride from human plasma samples. Plasma sample (900 microl) was deproteinated and extracted with ethanol and acetonitrile. The extract (70 microl) was directly injected into a Capcell Pak MF Ph-1 pre-column where the primary separation occurred to remove proteins and retain drugs using a mixture of acetonitrile and 10mM phosphate buffer (pH 2.18) (20:80, v/v). The analytes were transferred from the pre-column to an intermediate column using a switching valve and then subsequently separated on an analytical column and monitored with UV detection at 228 nm. Glimepiride was eluted with retention time 34.9 min without interference of endogenous substance from plasma. The limit of quantification (LOQ) was 10 ng/ml for glimepiride. The calibration curves were linear over the concentration range of 10-400 ng/ml (r(2) = 0.9997). Moreover, inter- and intra-day precisions of the method were less than 15% and accuracies were higher than 99%. The developed method was successfully applied for the quantification of glimepiride in human plasma and was used to support a human pharmacokinetic study following a single oral administration of 2 mg glimepiride.