Simultaneous determination of three corticosteroids (clobetasol propionate, betamethasone dipropionate, fluticasone propionate) in moisturizers was performed by using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Sample preparation was conducted by the liquid-liquid extraction (LLE). Moisturizers include emulsifying agent and it forms micelles. In order to improve the extraction efficiency of corticosteroids trapped in micelle, newly developed-optimized extraction conditions which can remove the matrix effect from moisturizers was applied with various pH conditions in LLE extraction stage of sample preparation. Thus, the addition of 10 μL of 1 M HCl into moisturizers sample before extraction could improve the extraction efficiency. For the quantitative analysis, SRM table that contained specific transition of all of target corticosteroids was created. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), limit of quantization (LOQ) and recovery. Over the 0.99 r2 value was obtained in calibration standard range. Effective accuracy and precision were also obtained. LODs were below 31 ng/mL and LOQs were estimated below 94 ng/mL for all corticosteroids tested.
An ultra-fast generic LC-MS/MS method was developed for high-throughput quantification of discovery pharmacokinetic(PK) samples and its reliability was verified. The method involves a simple protein precipitation for sample preparationand the analysis by ultra-fast generic LC-MS/MS with the ballistic gradient program and selected reaction monitoring (SRM)mode. Approximately 290 new chemical entities (NCEs) (over 10,000 samples) from 5 therapeutic programs were analyzed. The calibration curves showed good linearity in the concentration range of 1, 2 or 5 to 2000 ng/mL. No significant ion suppressionwas observed in the elution region of all the NCEs. When approximately 300 plasma samples were continuously analyzed,the peak area of internal standard was constant and reproducible. In the repeated analysis of samples, the plasma concentrationsand the area under the curve (AUC) were consistent with the results from the first analysis. These results showed that the presentultra-fast generic LC-MS/MS method is reliable in terms of selectivity, sensitivity, and reproducibility and could be useful forhigh-throughput quantification and other bioanalysis in drug discovery.
Dioscin is a biologically active steroidal saponin with anticancer and hepatoprotective effects. A rapid, selective, andsensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for the quantificationof dioscin in rat plasma. Dioscin was extracted from rat plasma using ethyl acetate at acidic pH. The analytes were separatedon a Halo C18 column using gradient elution of acetonitrile and 0.1% formic acid and detected by tandem massspectrometry in selected reaction monitoring mode. The standard curve was linear (r2 = 0.998) over the concentration range of1−100 ng/mL. The lower limit of quantification was 1.0 ng/mL using 50 μL of plasma sample. The coefficient of variation andrelative error for intra- and inter-assay at four QC levels were 1.3 to 8.0% and −5.4 to 10.0%, respectively. This method wasapplied successfully to the pharmacokinetic study of dioscin after oral administration of dioscin at a dose of 29.2 mg/kg in maleSprague-Dawley rats.
Coptidis Rhizoma (CR) has been used widely in traditional medicine to treat common diseases. This study aimed to develop a high-sensitivity liquid chromatography-tandem mass (LC-MS) spectrometry method for the evaluation of the pharmacokinetics of a new natural product that contain CR extract with the main bioactive compound, berberine, at trace concentrations. Human plasma samples were pretreated with methanol by a protein precipitation method. Berberine was analyzed on a Kinetex C18 column (2.1 mm × 50 mm, 100 Å, 1.7 µm) using a mobile phase of 10 mM ammonium formate/0.1% formic acid in water (A) and acetonitrile (B) (50:50, v/v) with a flow rate of 0.25 mL/min. The analyte was detected by using electrospray ionization in positive mode with multiple reaction monitoring (MRM). The method was sensitive, with a lower limit of quantification of 1 pg/mL, which has not been previously obtained. The method was validated (over the range of 1–50 pg/mL) and applied successfully for the pharmacokinetic study of human plasma samples.
Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processes for accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin digestion using an instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in the sample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate the effectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T), and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal tryptic digestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in the number of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed higher numbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis of heat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were compared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestion efficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.
Nonmedical use of prescription stimulants such as methylphenidate (MPH) and amphetamine (AP) by normal persons has been increased to improve cognitive functions. Due to high potential for their abuse, reliable analytical methods were required to detect these prescription stimulants in biological samples. A direct injection liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and implemented for simultaneous determination of MPH, AP and their metabolites ritalinic acid (RA) and 4-hydroxyamphetamine (HAP) in human urine. Urine sample was centrifuged and the upper layer (100 μL) was mixed with 800 μL of distilled water and 100 μL of internal standards (0.2 μg/mL in methanol). The mixture was then directly injected into the LC-MS/MS system. The mobile phase was composed of 0.2% formic acid in distilled water (A) and acetonitrile (B). Chromatographic separation was performed by using a Capcell Pak MG-II C18 (150 mm × 2.0 mm i.d., 5 μm, Shiseido) column and all analytes were eluted within 5 min. Linear least-squares regression with a 1/x weighting factor was used to generate a calibration curve and the assay was linear from 20 to 1500 ng/mL (HAP), 40-3000 ng/mL (AP and RA) and 2-150 ng/mL (MPH). The intra- and inter-day precisions were within 16.4%. The intra- and inter-day accuracies ranged from -15.6% to 10.8%. The limits of detection for all the analytes were less than 4.7 ng/mL. The suitability of the method was examined by analyzing urine samples from drug abusers.
The pharmacokinetic properties of S-amlodipine were studied using racemic amlodipine and single S-enantiomer(SK310) administration to rats. Plasma levels of the drug were determined using chiral liquid chromatography coupled with tandemmass spectrometry following solid phase extraction. The stereospecific analysis of amlodipine was performed on an α-acidglycoprotein (AGP) column using a mobile phase comprising 10 mM ammonium acetate (pH 4.0) and propanol at a flow rate of0.2 mL/min. This method was used to perform a comparative study of the pharmacokinetics of amlodipine and SK310. Theresults revealed that the pharmacokinetic profile of S-amlodipine after the administration of SK310 was comparable to thatfollowing the administration of the racemic mixture.
Arctigenin is the main active ingredient of Fructus Arctii, which has been reported with a variety of therapeutic activities including anti-cancer, anti-inflammation, anti-virus, and anti-obesity effects. In this study, a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of arctigenin in rat plasma. The assay utilized a simple protein precipitation with methanol and the mobile phase consisted of 100% methanol and water containing 0.1% formic acid (65:35 v/v). Arctigenin and the internal standard (psoralen) were monitored using a positive electrospray turbo ionspray mode with multiple reaction monitoring transitions of m/z 373.2→136.9 and m/z 187.2→130.9, respectively, and total chromatographic run time was within 5 min. The lower limit of quantification (LLOQ) of arctigenin was 5 ng/mL in the rat plasma. The intra- and inter-day accuracy of arctigenin at LLOQ and matrix-matched quality control samples ranged 97.4 – 104.8% and 97.2 – 102.0%, respectively. The intra-day precision was within 4.80% and the interday precision was within 5.92%. Application of the present method was demonstrated through a pharmacokinetic study after intravenous and oral administration of arctigenin in male Sprague Dawley rats.
Mertansine, a thiol-containing maytansinoid, is a tubulin inhibitor used as the cytotoxic component of antibody-drug conjugates for the treatment of cancer. Liquid chromatography-tandem mass spectrometry was described for the determination of mertansine in rat plasma. 50-µL rat plasma sample was pretreated with 25 µL of 20 mM tris-(2-carboxyethyl)-phosphine, a reducing reagent, and further vortex-mixing with 50 µL of 50 mM N-ethylmaleimide for 3 min resulted in the alkylation of thiol group in mertansine. Alkylation reaction was stopped by addition of 100 µL of sildenafil in acetonitrile (200 ng/mL), and following centrifugation, aliquot of the supernatant was analyzed by the selected reaction monitoring mode. The standard curve was linear over the range of 1–1000 ng/mL in rat plasma with the lower limit of quantification level at 1 ng/mL. The intra- and inter-day accuracies and coefficient variations for mertansine at four quality control concentrations were 96.7–113.1% and 2.6–15.0%, respectively. Using this method, the pharmacokinetics of mertansine were evaluated after intravenous administration of mertansine at doses of 0.2, 0.5, and 1 mg/kg to female Sprague Dawley rats.
We aimed to develop and validate a sensitive analytical method of nannozinone A, active metabolite of Nannochelins A extracted from the Myxobacterium Nannocytis pusilla, in mouse plasma using a liquid chromatography-tandem mass spec-trometry (LC-MS/MS). Mouse plasma samples containing nannozinone A and 13 C-caffeine (internal standard) were extracted using a liquid-liquid extraction (LLE) method with methyl tert-butyl ether. Standard calibration curves were linear in the concen-tration range of 1 - 1000 ng/mL (r 2 > 0.998) with the inter- and intra-day accuracy and precision results less than 15%. LLE method gave results in the high and reproducible extraction recovery in the range of 78.00–81.08% with limited matrix effect in the range of 70.56-96.49%. The pharmacokinetics of nannozinone A after intravenous injection (5 mg/kg) and oral administra- tion (30 mg/kg) of nannozinone A were investigated using the validated LC-MS/MS analysis of nannozinone A. The absolute oral bioavailability of nannozinone A was 8.82%. Plasma concentration of nannozinone A after the intravenous injection sharply decreased for 4 h but plasma concentration of orally administered nannozinone A showed fast distribution and slow elimination for 24 h. In conclusion, we successfully applied this newly developed sensitive LC-MS/MS analytical method of nannozinone A to the pharmacokinetic evaluation of this compound. This method can be useful for further studies on the pharmacokinetic opti-mization and evaluating the druggability of nannozinone A including its efficacy and toxicity.