Evaluation of absolute peptide quantitation strategies using selected reaction monitoring

The use of internal peptide standards in selected reaction monitoring experiments enables absolute quantitation. Here, we describe three approaches addressing calibration of peptide concentrations in complex matrices and assess their performance in terms of trueness and precision. The simplest approach described is single reference point quantitation where a heavy peptide is spiked into test samples and the endogenous analyte quantified relative to the heavy peptide internal standard. We refer to the second approach as normal curve quantitation. Here, a constant amount of heavy peptide and a varying amount of light peptide are spiked into matrix to construct a calibration curve. This accounts for matrix effects but due to the presence of endogenous analyte, it is usually not possible to determine the lower LOQ. We refer to the third method as reverse curve quantitation. Here, a constant amount of light peptide and a varying amount of heavy peptide are spiked into matrix to construct a calibration curve. Because there is no contribution to the heavy peptide signal from endogenous analyte, it is possible to measure the equivalent of a blank sample and determine LOQ. These approaches are applied to human plasma samples and used to assay peptides of a set of apolipoproteins.     

Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC): practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis

A selective, accurate, and reproducible LC/MS/MS assay was developed and validated for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) samples. In addition to the details of the validated LC/MS/MS method, a practical procedure is described in great detail for the preparation of large supplies of control (blank) PBMC from units of blood (each unit of blood is about 500 ml) for making the calibration standards and quality control (QC) samples. The PBMC assay design, intended for high-throughput sample analysis, is also described in some detail in regards to the composition and concentration expressions of the calibration standards and QC samples, the lysing procedure of the PBMC samples, and the final analysis/quantitation procedure. The method involved automated solid-phase extraction (SPE) of atazanavir and a stable isotope analog internal standard (I.S.) using 3M Empore C2-SD 96-well plates. A portion of the reconstituted sample residue was injected onto a YMC Basic analytical column which was connected to a triple quad mass spectrometer for analyte determination by positive-ion electrospray in the selected reaction monitoring (SRM) mode. The standard curve, which ranged from 5 to 2500 fmol per one million cells (fmol/10(6) cells), was fitted to a quadratic regression model weighted by 1/concentration. The lower limit of quantitation (LLOQ) was 5 fmol/10(6) cells. The inter- and intra-run coefficients of variation (CV) for the assay were <9% and the accuracy was 94-104%. Atazanavir was stable in PBMC for at least 24h at room temperature and for at least 129 days at -15 degrees C.     

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.     

Determination of glabridin in human plasma by solid-phase extraction and LC-MS/MS

Glabridin is a major flavonoid included specifically in licorice (Glycyrrhiza glabra L.), and has various physiological activities including antioxidant and anti-inflammatory effects. We have developed and validated an analytical method for determination of glabridin in human plasma by solid-phase extraction (SPE) and LC-MS/MS. Glabridin was extracted from plasma by SPE using a C8 cartridge and analyzed by LC-MS/MS using mefenamic acid as an internal standard (IS). The analyte were separated by a C18 column on LC, and monitored with a fragment ion of m/z 201 formed from a molecular ion of m/z 323 for glabridin and that of m/z 196 from m/z 240 for IS during negative ion mode with tandem MS detection. The lower limit of quantitation (LLOQ) of glabridin was 0.1 ng/mL in plasma, corresponding to 1.25 pg injected on-column. The calibration curves exhibited excellent linearity (r>0.997) between 0.1 and 50 ng/mL. Precision and accuracy were <17 and <+/-7% at LLOQ, and <11 and <+/-5% at other concentrations. Glabridin was recovered >90%, and was stable when kept at 10 degrees C for 72 h, at -20 degrees C until 12 weeks, and after three freeze-thaw cycles. This is the first report on determination of glabridin in body fluids by the selective, sensitive, and reproducible method.     

Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph) and sphingosine 1-phosphate (Sph-1P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at 1 pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.     

Ultra-fast quantitation of saquinavir in human plasma by matrix-assisted laser desorption/ionization and selected reaction monitoring mode detection

We present herein an ultra-fast quantitative assay for the quantitation of saquinavir in human plasma, without prior chromatographic separation, with matrix-assisted laser desorption/ionization using the selected reaction monitoring quantitation mode (MALDI-SRM/MS). The method was found to be linear from 5 to 10,000ng/ml using pentadeuterated saquinavir (SQV-d5) as an internal standard, and from 5 to 1000ng/ml using reserpine as internal standard (IS). Accuracy and precision were in the range of 101-108%, 3.9-11% with SQV-d5 and in the range 93-108%, 3.5-15% with reserpine. Plasma samples (250mul) were extracted with a mixture of ethyl acetate/hexane. MALDI spotting of the extract was automated using electrodeposition and the dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) as matrix. A 96 spots MALDI plate was prepared within 20min in a fully unattended manner. Each sample was spotted four times and quantitation was based on the average of their analyte/IS area ratio. Samples were analyzed on a triple quadrupole linear ion trap (QqQ(LIT)) equipped with a high repetition laser source (1000Hz). The analysis time of one sample was approximately 6s, therefore 96 samples could be analyzed in less than 10min. With liquid-liquid extraction sample preparation no significant matrix effects were observed. Moreover, the assay showed sufficient selectivity for samples to be analyzed at the lower limit of quantification (LLOQ) in the presence of other antiretroviral drugs, without prior chromatographic steps. In parallel, to assess the selectivity of the assay with real samples, a liquid chromatography (LC)-SRM/MS method was developed and a cross validation with clinical samples was successfully performed.     

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

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

Methodology for real-time,multianalyte monitoring of fermentations using an in-situ mid-infrared sensor

An in-situ, mid-infrared sensor was used to monitor the major analyte concentrations involved in the cultivation of Gluconacetobacter xylinus and the production of gluconacetan, a food-grade exopolysaccharide. To predict the analyte concentrations, three different sets of standard spectra were used to develop calibration models, applying partial least-squares regression. It was possible to build a valid calibration model to predict the 700 spectra collected during the complete time course of the cultivation, using only 12 spectra collected every 10 h as standards. This model was used to reprocess the concentration profiles from 0 to 15 g/L of nine different analytes with a mean standard error of validation of 0.23 g/L. However, this calibration model was not suitable for real-time monitoring as it was probably based on non-specific spectral features, which were correlated only with the measured analyte concentrations. Valid calibration models capable of real-time monitoring could be established by supplementing the set of 12 fermentation spectra with 42 standards of measured analytes. A pulse of 5 g/L ethanol showed the robustness of the model to sudden disturbances. The prediction of the models drifted, however, toward the end of the fermentation. The most robust calibration model was finally obtained by the addition of 34 standard spectra of non-measured analytes. Although the spectra did not contain analyte-specific information, it was believed that this addition would increase the variability space of the calibration model. Therefore, an expanded calibration model containing 88 spectra was used to monitor, in real time, the concentration profiles of fructose, acetic acid, ethanol and gluconacetan and allowed standard errors of prediction of 1.11, 0.37, 0.22, and 0.79 g/L, respectively.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Development and validation of a selective and sensitive LC-MS/MS method for determination of cycloserine in human plasma: application to bioequivalence study

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20-30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.     

A dilution immunoassay to measure myosin regulatory light chain phosphorylation

We examined the quantitation of myosin regulatory light chain phosphorylation (MRLCP) by Western blot and found both offset and saturation errors. The desirable characteristics of an MRLCP assay are that the dynamic range be 60- to 100-fold and that the detection threshold be known and preferably very small relative to total MRLC concentration. No technique examined provided all these characteristics. However, accurate measurements can be obtained by including serial dilutions of the sample to provide a fractional calibration scale in terms of the dephosphorylated light chain and by using interpolation of the phosphorylated band signal intensity to provide values for the relative phosphorylation ratio. We found that this method offers several advantages over methods that rely on signal ratios from single samples: The dilution ratio method is less subject to errors from differences in protein load, it offers estimates of the error in the individual measurement, and has some redundancy that increases the likelihood of obtaining a valid measurement despite gel or membrane artifacts.     

Validation of a gas chromatography-mass spectrometry method for the determination of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum

A method for the quantitation of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum by gas chromatography/electron-capture mass spectrometry has been developed and validated. Using the area ratios of the integrated molecular-ion peaks of the analytes to their corresponding deuterated internal standards, [2,4,16,16-2H4] 17beta-estradiol (17beta-estradiol-d(4)) and [16,16-2H2] 17beta-trenbolone (17beta-trenbolone-d(2)), and non-weighted linear regression, two calibration curves per analyte; 5-50 and 50-500 pg/ml for 17beta-estradiol in sera, and 25-250 and 250-2500 pg/ml for 17beta-trenbolone in sera, respectively, were constructed. Splitless injection of 200 fg 17beta-estradiol and 1000 fg 17beta-trenbolone could be detected and quantified. Tested batches of control bovine sera did not exhibit interference for 17beta-trenbolone, and showed expected background presence of endogenous 17beta-estradiol. Intra-day residual errors did not exceed 20%, and regression correlations were greater than 0.99. Intra-day precision data was similar to inter-day precision data. Using this method, 16 samples can be processed within one working day.     

Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.     

Quantitative determination of perfluorooctanoic acid in serum and plasma by liquid chromatography tandem mass spectrometry

A selective and sensitive method for analysis of perfluorooctanoic acid (PFOA) in human serum and plasma, utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS), has been developed and thoroughly validated to satisfy strict FDA guidelines for bioanalytical methods. A simple, automated sample preparation procedure, involving extraction of the target analyte with acetonitrile on protein precipitation media in a 96-well plate format was developed, allowing efficient handling of large numbers of samples. The proposed method uses the calibration standards prepared in a surrogate matrix (rabbit serum or plasma) and (13)C-labeled PFOA as the internal standard to account for matrix effects, instrument drift, and extraction efficiency. Human serum and plasma could not be used for matrix matching of calibration standards as endogenous levels of PFOA observed in the control human serum and plasma significantly exceeded the targeted lower limit of quantitation (LLOQ) of the method. Precision and accuracy of the method were demonstrated by analysis of rabbit serum and plasma control samples fortified at 0.5, 5, and 40 ng/mL PFOA and human serum and plasma fortified at 1.0, 5.0, 40 ng/mL PFOA. The LLOQ of 0.5 ng/mL PFOA was experimentally demonstrated for rabbit and human serum and plasma. Within-day precision and accuracy, short-term stability, freeze-thaw stability, equivalence of response between PFOA and APFO (the ammonium salt of PFOA), and dilution of concentrated samples were also investigated. The results of the validation experiments comply with the precision and accuracy limits defined by the FDA guidance document: "Guidance for Industry, Bioanalytical Method Validation", May 2001.     

Sensitive method for the quantitative determination of gemfibrozil in dog plasma by liquid-liquid cartridge extraction and liquid chromatography-tandem mass spectrometry

A sensitive LC-MS/MS assay for the quantitative determination of gemfibrozil in dog plasma has been developed and validated and is described in this work. The assay involved the extraction of the analyte from 0.5-ml aliquots of dog plasma using Chem Elut cartridges and methyl tert.-butyl ether (MTBE). Chromatography was performed on a Metasil Basic column (50 x 2 mm I.D., 3 microm) using a mobile phase that consisted of 70:30 acetonitrile-ammonium acetate (1 mM, pH 5.0) with a flow-rate of 0.2 ml min(-1). The method showed excellent reproducibility with an inter- and intra-assay precision of <8.9% (%RSD), as well as excellent accuracy with an inter- and intra-assay accuracy between 99 and 101%. This method has a lower limit of quantitation (LLOQ) of 1.0 ng ml(-1) with a linear calibration range from 1.0 to 250 ng ml(-1). This new assay offers higher sensitivity and a much shorter run time over earlier methods.     

Effect of different matrices on physiological amino acids analysis by liquid chromatography: evaluation and correction of the matrix effect

Free amino acids in plasma, muscle and liver tissues were determined using the Pico-Tag method which involves HPLC and pre-column derivatization. A study of the accuracy in the determination is carried out by means of external calibration, standard-added calibration, Youden calibration and analyte recovery. It is conclusively shown that the method is affected by a proportional systematic error due to matrix effects, but not by a constant one. A new function named matrix-corrected calibration is proposed to correct for proportional bias when free analyte matrices for matrix-matched addition is unavailable.     

Surrogate based accurate quantification of endogenous acetylcholine in murine brain by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Cholinergic dysfunction is known as a hallmark feature of Alzheimer's disease (AD). Measurement of endogenous acetylcholine (ACh) in specific brain regions is important in understanding the pathology of AD and in designing and evaluating novel cholinomimetic agents for the treatment of AD. Since ACh is an endogenous neurotransmitter, there is no real blank matrix available to construct standard curves. It has been a challenging task to determine ACh in complex brain matrices. To overcome these difficulties, we employed a surrogate analyte strategy using ACh-d(4) instead of ACh to generate calibration curves and Ch-d(9) as internal standard (IS). The brain samples were deproteinized by acetonitrile with IS. Analytes and IS were separated by a HILIC column with the mobile phase composed of 20 mM ammonium formate in water-acetonitrile (30:70, v/v, adjusted to pH 3.0 with formic acid) and monitored in multiple reaction monitoring (MRM) mode using a positive electrospray source. The concentrations of endogenous ACh were calculated based on the peak area ratio of the analyte to the IS using a regression equation for the corresponding surrogate standard (ACh-d(4)). The lower limit of detection was 0.2 ng/mL and linearity was maintained over the range of 10-1000 ng/mL. Compared to other currently available methods, this approach offers improved accuracy and precision for efficient analysis of ACh. The proposed method was proved successfully by evaluating the action of typical acetylcholinesterase inhibitor huperzine A in senescence accelerated mouse prone 8 (SAMP8).     

Low-level quantitation of oxycodone and its oxidative metabolites, noroxycodone, and oxymorphone, in rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry

A method was developed for quantification of oxycodone, noroxycodone, and oxymorphone in small volumes (50 microl) of rat plasma by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry using turbo ion-spray. Deuterated (d3) opioid analogues acted as internal standards. Sample preparation involved protein precipitation with acetonitrile, centrifugal evaporation, and reconstitution in mobile phase; analyte separation was performed on a C18 (5 microm, 2.1 mm x 50 mm) column using a linear gradient program. Lower limits of quantitation (ng/ml) and their between-day accuracy and precision were-oxycodone, 0.9 (-0.2 and 7.8%); noroxycodone, 1.0 (0.6 and 6.2%); oxymorphone 1.0 (-1.8 and 9.5%).     

Sensitive method for the determination of ibutilide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for determination of ibutilide in human plasma. The analyte and internal standard sotalol were extracted from plasma samples by liquid-liquid extraction, and separated on a C(18) column, using acetonitrile-water-10% butylamine-10% acetic acid (80:20:0.07:0.06, v/v/v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via TurboIonSpray ionization (ESI). Linear calibration curves were obtained in the concentration range of 20-10,000 pg/ml, with a lower limit of quantitation of 10 pg/ml. The intra- and inter-day precision values were below 8% and accuracy was within +/-3% at all three QC levels. The method was utilized to support clinical pharmacokinetic studies of ibutilide in healthy volunteers following intravenous administration.     

An HPLC method for the pharmacokinetic study of daidzein-loaded nanoparticle formulations after injection to rats

This study was aimed at developing a simple HPLC method for the detection of daidzein in rat plasma. Daidzein was extracted from rat plasma with ethylparaben as internal standards (IS). Chromatographic separation of daidzein and IS was achieved by a Dikma Dimonsil C18 column (200 mm × 4.6mm) with the mobile phase consisting of methanol-water (55:45, v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 μL and the detecting wavelength was 249 nm. The calibration curve was linear over a concentration range from 0.05 to 5 μg/mL, and the accuracy was within a range of 93.4-126.2%. This HPLC method was applied successfully to the pharmacokinetic study of two kinds of daidzein-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (D-NPs) and daidzein suspension after intravenous injection in rats. Significant differences in main pharmacokinetic parameters of daidzein suspension and D-NPs were observed.