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.     

High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometry for the detection of 17alpha-ethinylestradiol in hepatocytes

Atmospheric pressure photoionization (APPI) as an interface for the high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) system was employed for the direct determination of 17alpha-ethinylestradiol (EE(2)) in the incubation mixtures to support in vitro hepatic clearance studies. For the APPI source, the radical cation of the analyte via charge exchange with the dopant radical cation was used for the detection of EE(2) in the positive ion mode. It was demonstrated that the major signals of EE(2) in the acetonitrile/water mobile phase were substantially increased by replacing toluene with anisole as the dopant. The effects of several experimental conditions on the photoionization efficiency of EE(2) in the dopant-assisted APPI source were explored. Electrospray ionization (ESI) source was also suitable for the analysis of the analyte; however, ESI required a derivatization step prior to analysis. The applicability of the proposed HPLC-APPI-MS/MS approach following a protein precipitation procedure for the determination of EE(2) at low nano-mole levels was examined with respect to assay specificity and linearity. The assay results obtained by both HPLC-APPI-MS/MS and HPLC-ESI-MS/MS methods were in good agreement.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Simple and sensitive liquid chromatography-tandem mass spectrometry methods for quantification of paraquat in plasma and urine: application to experimental and clinical toxicological studies

Simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed and validated for quantification of paraquat (PQ) in plasma and urine. Plasma and urine sample preparation were carried out by one-step protein precipitation using cold acetonitrile (-20 to -10 °C). After centrifugation, an aliquot of 10 μL of supernatant was injected into a Kinetex? hydrophilic interaction chromatography (HILIC) column with a KrudKatcher? Ultra in-line filter. The chromatographic separation was achieved using the mobile phase mixture of 250 mM ammonium formate (with 0.8% aqueous formic acid) in water and acetonitrile at a flow rate of 0.3 mL/min. Detection was performed using an API2000 triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear over the concentration range of 10-5000 ng/mL, with an LLOQ of 10 ng/mL. The inter- and intra-day precision (% R.S.D.) were <8.5% and 6.4% for plasma and urine, respectively with the accuracies (%) within the range of 95.1-102.8%. PQ in plasma and urine samples was stable when stored at -70 °C for three freeze-thaw cycles. The methods were successfully applied to determine PQ concentration in rat and human samples.     

Sensitive method for the quantitative determination of proguanil and its metabolites in rat blood and plasma by liquid chromatography-mass spectrometry

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.     

Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 microL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 microL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3>541.3, 544.2>501.2, and 615.3>572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5-3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 microL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.     

Quantitative 2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted plasma

Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62+/-1.2% of the total protein, 93+/-1.4% of the albumin (0.43 microg/microL, residual), and 94+/-1.5% of the IgG (0.21 microg/microL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675-1325), with 10+/-6% inter-gel variability in spot density. LC-ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.     

Comparison of plasma and dry blood spots as samples for the determination of nitisinone (NTBC) by high-performance liquid chromatography-tandem mass spectrometry. Study of the stability of the samples at different temperatures

Tyrosinemia is an inborn error of metabolism characterized by the accumulation of tyrosine as well as toxic by-products. NTBC or nitisinone is a drug currently used for the treatment of tyrosinemia that avoids the formation of these toxic substances. This paper presents the determination of NTBC in plasma and dry blood spots by high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. The concentration of NTBC in matched plasma-dry blood spots was compared and the study of degradation of NTBC in plasma and dry spots at different temperatures is presented. Method: For sample preparation, plasma proteins were precipitated with acetonitrile and 3-mm discs were extracted with methanol. ESI(+) was used as inozation method and the analytes were detected by multiple reaction monitoring using the transitions 330>218 for NTBC and 340>228 for mesotrione, used as internal standard. Results: There is good correlation between concentrations obtained in dry blood spots and plasma (r(2)=0.83), although values are 2.4 times higher in plasma samples. NTBC in plasma is stable at least for 45 days frozen at -30°C and refrigerated at 4°C. However, it shows slow decomposition at room temperature, approximately 30% after 45 days. The method shows good precision, accuracy and linearity and the detection limit is 50 nmol/L and paper samples are appropriate for the monitorization of NTBC.     

Direct cocktail analysis of drug discovery compounds in pooled plasma samples using liquid chromatography-tandem mass spectrometry

Direct plasma injection technology coupled with a LC-MS/MS assay provides fast and straightforward method development and greatly reduces the time for the tedious sample preparation procedures. In this work, a simple and sensitive bioanalytical method based on direct plasma injection using a single column high-performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS) was developed for direct cocktail analysis of double-pooled mouse plasma samples for the quantitative determination of small molecules. The overall goal was to improve the throughput of the rapid pharmacokinetic (PK) screening process for early drug discovery candidates. Each pooled plasma sample was diluted with working solution containing internal standard and then directly injected into a polymer-coated mixed-function column for sample clean-up, enrichment and chromatographic separation. The apparent on-column recovery of six drug candidates in mouse plasma samples was greater than 90%. The single HPLC column was linked to either an atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI) source as a part of MS/MS system. The total run cycle time using single column direct injection methods can be achieved within 4 min per sample. The analytical results obtained by the described direct injection methods were comparable with those obtained by semi-automated protein precipitation methods within +/- 15%. The advantages and challenges of using direct single column LC-MS/MS methods with two ionization sources in combination of sample pooling technique are discussed.     

Simultaneous determination of berberine, palmatine and jatrorrhizine by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of coptis-evodia herb couple

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine berberine, palmatine and jatrorrhizine in rat plasma. After mixing with the internal standard (IS) tetrahydropalmatine, plasma samples were pretreated by protein precipitation with acetonitrile-methanol (1:2, v/v). Chromatographic separation was carried out on a C18 column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 1.0-250.0 ng/mL for all components. The intra- and inter-day precision values were less than 14.6% and the deviations were within +/-4.0%. The fully validated LC-MS/MS method has been successfully applied to the pharmacokinetic study of berberine, palmatine and jatrorrhizine in rat plasma after oral administration of coptis-evodia herb couple. Three peaks were observed in both individual and mean plasma-concentration curves of berberine, palmatine and jatrorrhizine, which may be attributed to distribution re-absorption and enterohepatic circulation.     

A New Method to Quantify Ifosfamide Blood Levels Using Dried Blood Spots and UPLC-MS/MS in Paediatric Patients with Embryonic Solid Tumours

Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z¹⁺ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100–10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7–19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q₂₅ 14.4–Q₇₅ 29.0) and 3.8 μmol/L (Q₂₅ 1.5–Q₇₅ 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.     

Sensitive and specific LC-ESI-MS/MS method for the determination of a styrylquinoline, BA011FZ041, a potent HIV anti-integrase agent, in rat plasma

A LC-MS/MS method was validated for the determination of BA011FZ041, a styrylquinoline derivative. After addition of BA011FZ055 as internal standard (IS), the method involved solid phase extraction (SPE), LC separation with an ether-phenyl column and quantification by MS/MS after positive ESI. The calibration curve, ranging from 1 to 500 ng/mL was fitted to a 1/x-weighted quadratic regression model. Lower limit of quantification (LLOQ) was 1 ng/mL using 100 microL of plasma. Intra- and inter-assay precision and accuracy values were within the regulatory limits. The method was successfully applied to the determination of BA011FZ041 in rat plasma and PBMCs after i.v. dosing.     

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([(2)H(3)(15)N] sorafenib) was achieved on a Waters X-Terra C(18) (150 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3-7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).     

Quantitation of five nevirapine oxidative metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A multiple-reaction-monitoring LC/MS/MS method for the analysis of nevirapine oxidative metabolites, 2-hydroxynevirapine, 3-hydroxynevirapine, 8-hydroxynevirapine, 12-hydroxynevirapine, and 4-carboxynevirapine, in human plasma was developed and validated. The metabolites were isolated from 50 microL heparinized plasma by enzymatic hydrolysis of the glucuronide conjugates to the free metabolite followed by protein precipitation with acetonitrile. Peaks were quantitated at 3.03 min for the 4-carboxynevirapine metabolite, at 3.72, 4.27, 5.27, and 5.73 min for the positional 2-hydroxynevirapine, 12-hydroxynevirapine, 3-hydroxynevirapine, and 8-hydroxynevirapine metabolites, respectively, and 2.30 min for the internal standard, pirenzepine. The assay was accurate and precise based on assay validation controls over the nominal range of 0.010-1.0 mg/L. The average accuracy at the lowest concentration quality control (QC) sample was 16% (difference from theoretical value) for 8-hydroxynevirapine, all others were closer to their known respective standards. Within- and between-day precisions were within 12% for quality control samples for all five metabolites. Repetitive thawing and freezing did not have an effect on any metabolite through a minimum of three cycles. Thawed samples, remaining in plasma for 4 h before extraction, were within 5% of theoretical value. Stability of the extracted samples on the autosampler at room temperature was evaluated for 48 h and was observed to be within 12% of a fresh analytical sample for 2-hydroxynevirapine and 3-hydroxynevirapine; other metabolites were within 6% of theoretical value. The utility of the analytical method was demonstrated using trough steady-state plasma samples collected from 48 patients in a hepatic impairment study.     

Multi-component plasma quantitation of anti-hyperglycemic pharmaceutical compounds using liquid chromatography-tandem mass spectrometry

Type-2 diabetes is a disorder characterized by disrupted insulin production leading to high blood glucose levels. To control this disease, combination therapy is often used. Hypoglycemic agents such as metformin, glipizide, glyburide, repaglinide, rosiglitazone, nateglinide, and pioglitazone are widely prescribed to control blood sugar levels. These drugs provide the basis for the development of a quantitative multianalyte bioanalytical method. As an example, a highly sensitive and selective multi-drug method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. This rapid, automated method consists of protein precipitation of 20 microL of plasma coupled with gradient HPLC elution of compounds using 10 mM ammonium formate buffer and 0.1% formic acid in acetonitrile as the mobile phases. MS/MS detection was performed using turbo ion spray in the positive ion multiple reaction monitoring (SRM) mode. A lower limit of quantitation (LLQ) in a range of 1.0-5.0 ng/mL was achieved for all analytes. The linearity of the method was observed over a 500-fold dynamic range. Drug recoveries ranged from 86.2 to 94.2% for all analytes of interest. Selectivity, sample dilution, intra-day and inter-day accuracy and precision, and stability assessment were evaluated for all compounds.     

Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

LC–MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma

A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150 mm × 4.6 mm, 3 μm) column using a gradient elution mode with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10 mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68–106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.     

Ferrocene-based Diels-Alder derivatization for the determination of 1alpha-hydroxyvitamin D3 in rat plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry

A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1alphaOHD(3) in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05-15 ng/mL) exhibited acceptable linearity (r>0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4-112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in ESI and applicable for biological samples.     

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract

We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.