An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.     

An accurate quantitative LC/ESI-MS/MS method for sirolimus in human whole blood

Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI-MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI-MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5-50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI-MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.     

Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.     

Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS: performance and comparison with LC/MS/MS methods

We present a comprehensive, sensitive, and highly specific negative ion electrospray LC/MS method for identifying all structural classes of glucosinolates in crude plant extracts. The technique is based on the observation of simultaneous maxima in the abundances of the m/z 96 and 97 ions, generated by programmed cone voltage fragmentation, in the mass chromatogram. The abundance ratios lie in the range 1:2-1:4 ([m/z 96]/[m/z 97]). Examination of the corresponding full-scan mass spectra allows individual glucosinolates of all structural classes to be identified rapidly and with confidence. The use of linearly programmed cone voltage fragmentation enhances characteristic fragment ions without compromising the abundance of the analytically important [M - H]- ion and its associated (and analytically useful) sulfur isotope peaks. Detection limits are in the low nanogram range for full-scan, programmed cone voltage spectra. Comparison of the technique with LC/MS/MS methods (product ion, precursor ion, and constant neutral loss scans) has shown that the sensitivity and selectivity of the programmed cone voltage method is superior. Data obtained on a variety of plant extracts confirmed that the methodology was robust and reliable.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

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.     

Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.     

Identification and elimination of ion suppression in the quantitative analysis of sirolimus in human blood by LC/ESI-MS/MS

Ion suppression can negatively affect the performance characteristics of LC/ESI-MS/MS based methods, and we wished to identify sources of ion suppression in an assay for quantitating sirolimus in human whole blood. We first compared the peak areas of sirolimus and ascomycin added to human blood samples treated with and without extraction using octadecyl silyl (ODS)-silica gel after protein precipitation, and we found that water-soluble compounds cause the ion suppression for both drugs. Post-column infusion studies indicated that compounds retained in the sample after ODS extraction and protein precipitation caused ion suppression. MS analysis of these compounds suggested they were hydroxyl group-possessing phosphocholines, and this was confirmed using purified lysophosphatidylcholine variants. Therefore, we included a HybridSPE treatment step after the ODS extraction into the preanalytical workflow to remove phosphocholines, and this successfully eliminated the observed ion suppression for determining sirolimus concentration in human whole blood by LC/ESI-MS/MS. Sirolimus is a highly lipophilic molecule, and this study demonstrates the impact that preanalytical extraction and purification steps can have on a laboratory's ability to accurately detect and quantitate this and other lipophilic drugs.     

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.     

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.     

复合季铵盐消毒剂含量的测定与滴定误差分析

研究复合季铵盐消毒剂含量的测定与滴定误差分析。探讨了四苯硼钠标准液配制及对滴定分析终点颜色的影响,并与单季铵盐定量分析方法进行了对比。结果表明:四苯硼钠两相滴定法适合于复合季铵盐定量分析,但要注意四苯硼钠标准液配制的正确性及滴定终点颜色的判断。     

Processing methods for differential analysis of LC/MS profile data

Background  

Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

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.     

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells

Purinergic Signalling - Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinson’s disease (PD) progression. However, the identification...