液相色谱-串联质谱法同时测定大鼠血浆中阿霉素和塞来昔布

目的:建立同时测定大鼠血浆中阿霉素和塞来昔布的液相色谱-串联质谱(LC/MS/MS)方法,研究这两种药物联合应用的药代动力学.方法:大鼠尾静脉注射阿霉素和塞来昔布,眼眶取血并抗凝,离心分离血浆,采用乙酸乙酯提取血浆中的阿霉素和塞来昔布,N2吹干乙酸乙酯,残留物用50μL甲醇溶解,取20μL用于LC/MS/MS分析.结果:用LC/MS/MS法检测大鼠血浆中阿霉素和塞来昔布的线性范围为1-800ng/mL,日内、日间精密度(RSD)均小于15%,检测血浆低、中、高三个浓度(8、50、500ng/mL)阿霉素的回收率分别为101.2%、95.1%和91.4%,检测血浆低、中、高三个浓度(8、50、500ng/mL)塞来昔布的回收率分别为105.6%、106.8%和93.7%.大鼠尾静脉注射5.8mg/kg阿霉素和3.8mg/kg塞来昔布的半衰期分别为2.3 h和3.6h,曲线下面积分别为670 ng·h·mL-1和1480ng·h·mL-1.结论:建立的方法灵敏、准确、快速,适甩于阿霉素和塞来昔布的药代动力学研究.     

Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.     

Development and validation of an LC/MS/MS assay for mycophenolic acid in human peripheral blood mononuclear cells

The aim was to develop a LC/MS/MS method able to quantify mycophenolic acid (MPA) in the peripheral blood mononuclear cells (PBMCs) of transplanted patients. PBMCs were isolated from blood by a density gradient separation. The chromatographic separation was carried out on a Zorbax Stable Bond CN, 150 mmx2.1 mm, and MS/MS detection was performed after positive electrospray ionisation of the protonated parent ion. The calibration range was from 0.25 to 100 ng/sample. Extraction from the cells and ionisation recoveries reached 73.5 and 37.9%, respectively. Inaccuracy was always <10% with CVs<15%. MPA was stable at room temperature in the autosampler over 48 h and at -20 degrees C over 1.5 months. Application to clinical samples taken from patients treated with mycophenolate mofetil indicated that the method is suitable for measuring intracellular MPA.     

A sensitive assay for simultaneous determination of plasma concentrations of valganciclovir and its active metabolite ganciclovir by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm x 2.1mm, 5 microm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 microg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.     

Simultaneous quantitation of perfluoroalkyl acids in human serum and breast milk using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

A high throughput analytical method using a column switching high-performance liquid chromatography combined with isotope dilution tandem mass spectrometry (column switching-HPLC–MS/MS) was developed to simultaneously quantitate the concentrations of 7 perfluoroalkyl acids (PFAAs) in serum and 3 PFAAs in breast milk samples. The sample preparation includes addition of the isotope-labelled internal standard solution to breast milk and serum, enzymatic hydrolysis and filtration of milk samples, precipitation of proteins and analysis by column switching-HPLC–MS/MS. The limits of quantitation ranged from 0.1 to 0.4 μg/l for serum and 0.02 to 0.15 μg/l for breast milk samples. The method accuracies ranged between 73.2% and 100.2% for the different analytes at two concentrations in PFAAs spiked samples. The validity of the method was confirmed by analysing 20 serum and 20 breast milk samples.     

Sensitive quantification of sirolimus and everolimus by LC–MS/MS with online sample cleanup

Sirolimus and its derivative everolimus are widely used today as immunosuppressive agents for example in the transplantation medicine. The problematic pharmacokinetic behavior of those substances makes therapeutic drug monitoring mandatory. Therefore, a fast, simple and sensitive high-throughput procedure using online extraction with turbulent flow chromatography for the concurrent measurement of sirolimus and everolimus has been developed. 200 μl of whole blood was mixed with internal standard (23-desmethoxyrapamycin) and the precipitation solution and centrifuged. An aliquot of the supernatant was transferred into autosampler vials. 50 μl of the supernatant was injected into the LC system, where the analytes were extracted using turbulent flow chromatography and thereafter analyzed using reversed phase chromatography. Detection was done by atmospherical pressure chemical ionization (APCI) mass spectrometry in the negative ionization mode. The method has been fully validated and compared to a previously used method. The method was shown to be linear over the entire calibration range (2.2–43.7 μg/l for everolimus and 2.9–51.2 μg/l for sirolimus). The lower limit of quantification was 0.5 μg/l for both compounds. For within-day and between-day analysis, the CV's were <7.6% for everolimus and <8.7% for sirolimus, respectively. The accuracy was between 92.1% and 105% for everolimus and 96.1% and 106% for sirolimus. Recovery ranged between 46.3% and 50.6% for everolimus and 51.2% and 57.2% for sirolimus. The method was demonstrated to be free of matrix effects and comparable to the previously used method. The presented LC–MS/MS method, using turbulent flow chromatography online extraction, allows a fast, simple and reliable determination of everolimus and sirolimus.     

Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm × 2.0 mm, 3.0 μm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4 → 765.4 (m/z) for vincristine and 811.4 → 751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.     

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 μL) were subjected to deproteinization with acetonitrile (200 μL) and then, after centrifugation, 150 μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb^® 5 μm CNRP 4.6 × 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.     

Development of a liquid chromatography-mass spectrometry (LC/MS) assay method for the quantification of PSC 833 (Valspodar) in rat plasma

A liquid chromatography-mass spectrometry (LC/MS) assay method was developed for the quantification of PSC 833 in rat plasma, using amiodarone as internal standard (IS). Separation was achieved using a C(8) 3.5 microm (2.1 mm x 50 mm) column heated to 60 degrees C with a mobile phase consisting of acetonitrile-ammonium hydroxide 0.2% (90:10 v/v) pumped at a rate of 0.2 mL/min. Detection was accomplished by mass spectrometer using selected ion monitoring (SIM) in positive mode. An excellent linear relationship was present between peak height ratios and rat plasma concentrations of PSC 833 ranging from 10 to 5000 ng/mL (R(2)>0.99). Intra-day and inter-day coefficients of variation (CV%) were less than 15%, and mean error was less than 10% for the concentrations above the limit of quantification. The validated limit of quantification of the assay was 10 ng/mL based on 0.1 mL rat plasma. The method limit of detection, based on an average signal-to-noise (S/N) ratio of 3, was found to be 2.5 ng/mL. The assay was capable of measuring the plasma concentrations of PSC 833 in rats injected with a single dose of 5 mg/kg of the drug. PSC 833 and IS eluted within 4 min, free of interfering peaks. The method was found to be fast, sensitive, and specific for the quantification of PSC 833 in rat plasma.     

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.     

Monitoring of lopinavir and ritonavir in peripheral blood mononuclear cells, plasma, and ultrafiltrate using a selective and highly sensitive LC/MS/MS assay

For the determination of the HIV protease inhibitors lopinavir and ritonavir in human plasma, plasma ultrafiltrate, and peripheral blood mononuclear cells (PBMCs) a highly sensitive and selective method has been developed, validated, and applied to samples of a healthy volunteer. BD Vacutainer CPT and Amicon Centriplus centrifugal filter devices were used for separation of PBMCs and for ultrafiltrate generation, respectively. After liquid/liquid-extraction extracts were chromatographed isocratically within 6 min on a Jupiter Proteo column. The drugs were quantified using 2H5-saquinavir as internal standard and electrospray tandem mass spectrometry in the selected reaction monitoring mode. Limits of quantification for both analytes were 4.0 ng/mL in plasma, 0.2 ng/mL in ultrafiltrate, and 0.1 ng/cell pellet (approximately 3 x 10(6) cells) in PBMCs. The calibration ranges were linear over more than three logs with an over-all accuracy varying between 98.7% and 111.5% and an over-all precision ranging from 6.2% to 14.0% (SD batch-to-batch). After a regular oral dose of Kaletra (400 mg lopinavir, 100 mg ritonavir) analyte concentrations were detectable over a full dosing interval in plasma, ultrafiltrate, and PBMCs. The method is well suited for monitoring of free and total plasma, and intracellular lopinavir/ritonavir concentrations in samples from clinical trials.     

Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.     

Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation

Nitric oxide (NO) is synthesized by NO synthase from L-arginine, which can be competitively blocked by endogenous inhibitors such as asymmetric dimethylarginine (ADMA), but not by symmetric dimethylarginine (SDMA). ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH) to dimethylamine and citrulline. A growing number of published clinical studies documented a strong correlation between increased ADMA blood levels and cardiovascular morbidity and mortality. We present here a highly sensitive method for the determination of this compounds in plasma and urine by means of HPLC-MS. The sample preparation is very simple and comprises only protein precipitation and concentration in the case of plasma samples and dilution in the case of urine. The samples are derivatized automatically with orthophthaldialdehyde and 2-mercaptoethanol, are separated on a 250 mm x 4 mm RP18 column by gradient elution with formate buffer/methanol and are detected by ESI-MS. The calibration functions are linear and cover the range from normal to pathologic concentration values of the analytes. The intra-day relative standard deviation (R.S.D.) of the assay for ADMA in plasma is 7.5% and the corresponding inter-day R.S.D. is 5.7%. In urine, these values for ADMA are 3.8 and 6.4%, respectively. All other analytes in plasma as well as in urine exhibit intra-day R.S.D. below 8%. The corresponding inter-day R.S.D. are all below 13%.     

Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.     

Determination of metformin in plasma by capillary electrophoresis using field-amplified sample stacking technique

A capillary electrophoresis method was described for the determination of metformin in human plasma based on the extraction of the ion-pair with bromothymol blue into chloroform. Phenformin was used as internal standard. Field-amplified sample stacking injection was employed with an electrokinetic injection voltage of 10 kV for 10 s. The running buffer was 0.1 M phosphate buffer (pH 2.5), running voltage was 20 kV and the UV absorbance detection was set at 195 nm. The limit of quantitation was 0.25 μg/ml. Linearity range of calibration curve was 0.25 to 3.5 μg/ml. Recoveries for three levels (0.25, 1 and 2 μg/ml) were 80.24%, 67.44% and 58.97% (n=5 for each level), respectively. The intra-day precisions for the three levels were 11.9%, 3.09% and 4.33% and the inter-day precisions were 12.4%, 4.57% and 4.94%, respectively. The concentrations of metformin hydrochloride in human plasma of eight volunteers were measured after orally administrating metformin enteric-capsule and tablet.