Quantification of human serum albumin by combining chymotrypsin/trypsin digestion coupled with LC-MS/MS technique.

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05-1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from -3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples.

Unraveling the in vivo biological fate of mPEG2000-PDLLA2500-COOH diblock copolymers by LC-MS/MS based on CID in source technique.

Methoxy poly (ethylene glycol)-poly(D, L-lactic acid) (mPEG-PDLLA) is a biocompatible and amphiphilic diblock copolymer composed of a hydrophilic poly(ethylene glycol) block and a hydrophobic poly(D, L-lactic acid) block, which can self-assemble into micelles in aqueous solution. It is one of the most widely used diblock copolymers for drug delivery, drug solubilization and drug encapsulation. Fully characterizing the in vivo fate of mPEG-PDLLA diblock copolymers is important to promote the further development of polymer-based nanocarrier drug delivery systems. However, to date, a bioanalysis assay for simultaneous quantification of mPEG-PDLLA and mPEG has not been reported. In this study, we developed such a novel LC-MS/MS assay based on CID in source technique and used it to study the multiple-dose pharmacokinetic, tissue distribution and excretion of mPEG2000-PDLLA2500-COOH and mPEG2000 in rat after intravenous administration. The results indicate that mPEG2000-PDLLA2500-COOH and mPEG2000 are mainly distributed to the liver, lung, spleen and kidney after intravenous administration. mPEG2000-PDLLA2500-COOH is mostly excreted via the renal route in the form of mPEG2000. Overall, the results of this study provide a comprehensive and clear picture of the in vivo fate of mPEG2000-PDLLA2500-COOH which will be useful in evaluating the efficiency and safety of polymer-based nanocarrier drug delivery systems.

Development and validation of an LC-MS/MS assay with multiple stage fragmentation for the quantification of alachlor in McF-7 cells.

Alachlor is one of the most widely used herbicides and can also be a carcinogenic compound. It is of great significance to establish a sensitive analytical method for the determination of alachlor in the environment and organisms. In this study, a high-performance liquid chromatography tandem mass spectrometry cubed (LC/MS3) method was developed and validated to quantify alachlor in human breast cancer cells (McF-7 cells). The cell samples were processed by simple protein precipitation with acetonitrile, then the analytes were separated on a Waters AcQuity® UPLC BEH (2.1 × 50 mm I.D, 1.7 µm) column using the gradient elution with solvent A (0.1 % formic acid) and solvent B (acetonitrile) at a flow rate of 0.5 mL/min. MS3 detection in positive ion mode was used to detect the analytes. The MRM3 transitions at m/z 270.1 â†’ 238.0 â†’ 162.1 and 312.2 â†’ 238.1 â†’ 147.2 were used to determine alachlor and butachlor, respectively. The run time for each sample was only 4 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, lower limit of quantitation (LLOQ), etc. The LC/MS3 assay was linear in the concentration range 0.5-50 ng/mL (R2 ≥ 0.995). For all concentrations, the precision is < 9.49 %, and the intra-day and intra-day accuracy is < 13.05 %. Cytotoxic potential of alachlor against McF-7 cell lines was measured by MTT method after 48 h of incubation. For alachlor, half maximal inhibitory concentration (IC50) on McF-7 cells was 87.95 µg/mL. This method was successfully applied to cellular pharmacokinetic study of alachlor in McF-7 cells after administration with a dose of 20 µg/mL.

Cellular toxicity and pharmacokinetic study of butachlor by ultra-performance liquid chromatography-tandem mass spectrometry based on tandem mass spectrometry cubed technique.

Butachlor is an aromatic amide compound that plays a role as a herbicide, a xenobiotic, and an environmental contaminant. The aim of this work was to develop a highly selective and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method based on the tandem mass spectrometry cubed technique to determine butachlor in a biological matrix. Butachlor and internal standard acetochlor were separated on a Waters Acquity ultra-performance liquid chromatography BEH C18 column (2.1 × 50 mm, 1.7 µm) with gradient elution using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases. The transitions selected for tandem mass spectrometry cubed quantitative analysis in positive ion mode were: for butachlor, mass-to-charge ratio 312.2→238.1→162.1; for acetochlor, mass-to-charge ratio 270.1→224.0→148.1. The total running time for each sample was 5.5 min. The ultra-performance liquid chromatography-tandem mass spectrometry cubed method showed a linear relationship (R2 ≥ 0.995) in the concentration range of 0.5-100 ng/ml. The intra and interday accuracies are within the range of -10.6%-4.3% and precisions are between 4.48% and 13.14%. The novelty of the method is the use of tandem mass spectrometry cubed scanning mode, which improves selectivity and sensitivity. The results indicated that butachlor was cellular toxic. The safety of butachlor should be considered when it is used as a herbicide.

Quantitative analysis of polypropylene glycol polymers by liquid chromatography tandem mass spectrometry based on collision induced dissociation technique.

Polypropylene glycol (PPG) is a commonly used synthetic polymer in many fields. Investigating the toxicity and pharmacokinetic behavior of PPG polymers is necessary and important for evaluating their safety in medicine and daily cosmetics. In this study, PPG425, PPG1K and PPG2K were selected as the target polymers for cytotoxicity and cellular pharmacokinetics study of PPG polymers. Structural diversity and polydisperse molecular weights (MWs) are significant challenges for quantification of PPG polymers by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collision induced dissociation in source or collision cell generated a series of PPG-related product ions at m/z 59.0, 117.1, 175.1, 233.2, 291.2, 349.3, 407.2, 465.3 and 523.5 corresponding to fragments containing 1, 2, 3, 4, 5, 6, 7, 8, 9 repeating propylene oxide subunits. PPG425 was determined by the sum of the MRM acquisitions used the transitions [M+H]+1 precursor ions â†’ product ions. PPG1K and PPG2K were determined by the MRM acquisitions used the transitions [M+H]+1 precursor ions â†’ product ions at m/z 233.2(four subunits)→59.0(one subunit). Based on the collision induced disassociation technique and structural specific product ions, pharmacokinetic studies of PEG425, PPG1K and PPG2K were successfully conducted in McF-7 cells. The experimental results revealed that PPG polymers are not biologically inert and they can enter into McF-7 cells. The safety of PPG polymers should be considered when they are used as pharmaceutical or cosmetic excipients.

Ultrafast and high-throughput quantitative analysis of carbamazepine in human plasma by direct analysis in real time tandem mass spectrometry coupled with solid phase extraction to eliminate matrix effects.

Therapeutic drug monitoring of carbamazepine is necessary for its clinical application with the purpose to reach therapeutic concentration and reduce the risk of concentration-dependent toxicity. An ultrafast analytical assay for quantification of carbamazepine in human plasma was developed and validated based on direct analysis in real time tandem mass spectrometry (DART-MS/MS). After reversed phase solid phase extraction with Waters Oasis HLB, carbamazepine and internal standard carbamazepine-D2N15 were monitored by positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 237.1→194.0 and 240.1→196.2, respectively. The DART-MS/MS method is ultrafast and high-throughput and the analytical time for each sample is only 0.4 min. The assay was linear in the concentration range 0.50-30 µg/mL and intra- and inter-day accuracies were within ± 15% and trueness were < 13.9% at all concentrations. The method was successfully applied to therapeutic drug monitoring of carbamazepine in human plasma.