Selective determination of total normal microcystin by colorimetry, LC/UV detection and/or LC/MS

Microcystins, hepatotoxic cyclic heptapeptides, are produced by freshwater cyanobacteria, and are classified four groups according to the amino acid structure at unit 7. Normal microcystins contain N-methyldehydroalanine (Mdha) or dehydroalanine (Dha) at unit 7, and command the great part of all microcystins. As unusual microcystin classes, [Dhb⁷]microcystins, [ - and -Ala⁷, or N-MeAla⁷]microcystins and [ -Ser⁷]microcystins have been found.

On tumor initiation and/or promotion activities of microcystins, the tumor promotion activity of normal microcystins has been found, but cancer-related activities of microcystins belonging in the other classes have not been clear.

To determine normal microcystins as hepatotoxic tumor promoters, a selective determination method was developed. Only Mdha or Dha in normal microcystins was reacted with glutathione (GSH). The GSH-normal microcystins conjugates were reacted with trinitrobenzene sulfonate (TNBS). The TNB–GSH-normal microcystin conjugate can be determined as the total normal microcystin by colorimetry. After methanolysis of the conjugate, dimethyl TNB–glutamate from the conjugate was determined by liquid chromatography/ultraviolet detection (LC/UV) and/or liquid chromatography/mass spectrometry (LC/MS). The detection limits of the total normal microcystin by colorimetry, LC/UV and/or LC/MS were 1 μg, 10 and 0.1 ng, respectively.     

Determination of reduced folates in tumor and adjacent mucosa of colorectal cancer patients using LC‐MS/MS

A liquid chromatography electrospray ionization tandem mass spectrometry (LC‐MS/MS) method has been developed for the determination of 5,10‐methylenetetrahydrofolate (methyleneTHF), tetrahydrofolate (THF) and 5‐methyltetrahydrofolate (methylTHF) in colorectal mucosa and tumor tissues. The folate extraction method includes homogenization, heat and folate conjugase treatment to hydrolyze polyglutamyl folate to monoglutamyl folate. Before analysis on LC‐MS/MS, simple and fast sample purification with ultrafiltration (molecular weight cut‐off membrane, 10 kDa) was performed. Folates were detected and quantified using positive electrospray. The method described in the present paper was successfully applied to determine the level of three folate monoglutamates in mucosa and tumor samples from 77 colorectal cancer patients, starting from a limited amount of tissue. The results showed that the LC‐MS/MS method has a great advantage over other previously used methods because of its high sensitivity and selectivity. Significantly higher levels of methyleneTHF and THF were found in tumor compared with matched mucosa tissues. Folate levels in adjacent mucosa were associated with tumor location, age and gender. The correlation between folate levels and tumor site further strengthens the fact that development of right‐ and left‐sided tumors follows different pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a precise quantitative method for monitoring sirolimus in whole blood using LC/ESI–MS/MS

Sirolimus is used on patients after solid organ transplantation and on lymphangioleiomyomatosis (LAM) patients, and therapeutic drug monitoring is required in clinical practice. We have previously reported an accurate method for quantitative determination of sirolimus, but its sample preparation step was complicated. In this study, we developed a modified liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) method for sirolimus quantification. A supported liquid extraction cartridge was used to purify sirolimus from whole blood and ion suppression was mostly prevented. The validation results met the acceptance criteria. This method was compared with the antigen conjugated magnetic immunoassay (ACMIA) and our previously reported method, using whole blood samples from LAM patients. Comparison of the Bland–Altman plots of the currently developed method and the previous method revealed no significant difference between the two methods (mean bias, −2.02%; 95% CI, −7.81–3.78). The values obtained using ACMIA were significantly higher than those obtained using the current method by 13.87% (95% CI, 6.49–21.25) owing to cross-reactivity. The degrees of cross reactivities in LAM patients and in organ transplant patients were similar, and our LC/ESI–MS/MS method precisely measured the blood concentrations of sirolimus.     

A single liquid chromatography–quadrupole time‐of‐flight mass spectrometric method for the quantification of total antibody,antibody‐conjugated drug and free payload of antibody–drug conjugates

A single hybrid affinity‐captured‐LC‐TOF‐MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine–citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65–613.00 ng/mL with an equation y = ax² + bx + c for the antibody‐conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on‐bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00–100.00 μg/mL with an equation y = ax² + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC‐TOF‐MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01–2200 ng/mL with an equation y = ax² + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity‐captured‐LC‐TOF‐MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine–citrulline linker at the early drug discovery stage.     

Combined usage of cascade affinity fractionation and LC‐MS/MS for the proteomics of adult mouse testis

In this report, the proteomics of adult mouse testis were analyzed by the combined usage of cascade affinity fractionation and LC‐MS/MS. The differences between the selected affinity ligands in size, shape, structure, and biochemical characteristics, result in each ligand exhibiting a specific affinity to some protein groups. Therefore, a cascade composition of different ligands can be applied to the fractionation of complex tissue proteins. Ultimately, the fractions collected from cascade affinity fractionation were analyzed by LC‐MS/MS, which resulted in high confidence identification of a total of 1378 non‐redundant mouse testis protein groups, over 2.6 times as many proteins as were detected in the un‐fractionated sample (526). All detected proteins were bioinformatically categorized according to their physicochemical characteristics (such as relative molecular mass, pI, grand average hydrophobicity value, and transmembrane helices), subcellular location, and function annotation. This approach highlighted the sensitivity of this method to a wide variety of protein classes. Utilizing a combination of cascade affinity fractionation and LC‐MS/MS, we have established the largest proteomic database for adult mouse testis at the present time.     

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.     

Determination of telmisartan in human blood plasma: Part II: Liquid chromatography-tandem mass spectrometry method development, comparison to immunoassay and pharmacokinetic study

A new liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) method with on-line sample clean-up for the determination of telmisartan in human blood plasma is presented. This technique is compared to a previously introduced enzyme-linked immunosorbent assay (ELISA), where fluorescence is used as detection method. For the LC/MS method applying an internal calibration via a deuterated internal standard, the limit of detection was 0.3 ng/mL, the limit of quantification was 0.9 ng/mL and the linear range extended from 0.9 to 1000 ng/mL. Forty-eight plasma samples from four healthy volunteers were analyzed in a pharmacokinetic study to obtain data for the method comparison. As a result, these two new and independent analytical methods for the determination of telmisartan in human blood plasma proved to yield comparable results for the amount of analyte.     

Simultaneous quantitation of 15 bioactive components in Yupingfeng granules by LC–MS/MS

Yupingfeng granules (YPFG) is commonly used in the treatment of immunological diseases, inflammations, and pulmonary diseases. Several studies have found that chromones, flavones, and saponins were the major bioactive compounds of YPFG. However, few studies have reported accurate quantification methods of these compounds. This study aimed to establish a simple and rapid method by using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine 15 bioactive compounds in YPFG. The experimental parameters including extraction methods, extraction solvents, extraction time, solid–liquid ratio, and LC–MS/MS condition were optimized. The linearity, precision, repeatability, stability, and recovery of the established method were evaluated. The contents of 15 bioactive compounds in seven batches of YPFG samples were analyzed by the established method and the results were compared with the values determined by HPLC. The optimal extraction condition was to extract 0.1 g of YPFG by ultrasound with 50 mL 50% ethanol for 30 min. A Waters ACQUITY UPLCBEH C18 column using the 0.1% formic acid water solution and acetonitrile as mobile phase with a gradient elution was applied to the chromatographic separation. The linearity, precision, repeatability, stability, and recovery of the method were within acceptable ranges. Compared with HPLC analysis methods in Chinese Pharmacopoeia and literature, the established method was faster, simpler, more accurate, and more reliable. The method of simultaneous determination of 15 components in YPFG by LC–MS might provide a basis for the study of the bioactive compounds and the improvement of the quality standard of YPFG.     

Fast LC/MS in the analysis of small molecules

Liquid chromatography-mass spectrometry (LC/MS) has become one of the most widely used analytical techniques in both qualitative and quantitative analysis of small molecules. Recently, with the increasing demand for ever-higher sample throughput, the use of faster chromatographic separations has become popular, along with other LC/MS methods that decrease analytical cycle-time. The burgeoning use of LC/MS has meant that the primary expertise of many practitioners today is not in the field of LC/MS, which has been facilitated by the ease-of-use of modern LC/MS systems. An examination of the current state of the literature, relating to "fast LC/MS", should serve well to those new to LC/MS, and should help them in the development of fast LC/MS methods that are effective in terms of both the chromatography and the utilization of the mass spectrometer. This review paper focuses on fast LC/MS analyses of small molecules that have been reported in peer-reviewed publications.     

Extraction protocol and mass spectrometry method for quantification of doxorubicin released locally from prodrugs in tumor tissue

The localized conversion of inactive doxorubicin prodrug chemotherapeutics to pharmacalogically active forms is difficult to quantify in mouse tumor models because it occurs only in small regions of tissue. The tumor tissue extraction protocol and LC–MS/MS analysis method described here were optimized to obtain a detection limit of 7.8 pg for the activated doxorubicin and 0.36 ng for the doxorubicin prodrug. This method can be useful for determining the biodistribution and activation efficiency for many different doxorubicin prodrugs. It can also be used for quantification of doxorubicin from tumor models that have poor vascularization resulting in low tissue accumulation. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous LC–MS/MS analysis of eicosanoids and related metabolites in human serum,sputum and BALF

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC–MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra‐ and inter‐day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC–MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials.     

A validated LC–MS/MS method for quantitative determination of L-dopa in Fagioli di Sarconi beans (Phaseolus vulgaris L.)

An analytical method based on ultrasound assisted extraction (UAE) and liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI/MS/MS) was validated and applied for determining L-dopa in four ecotypes of Fagioli di Sarconi beans (Phaseolus vulgaris L.), marked with the European label PGI (Protected Geographical Indication). The selectivity of the proposed method was ensured by the specific fragmentation of the analyte. Simple isocratic chromatographic conditions and mass spectrometric detection in multiple reaction monitoring (MRM) acquisition mode were used for sensitive quantification. The LC–ESI/MS/MS method was validated within a linear range of 0.001–5.000 μg/mL. Values of 0.4 and 1.1 ng/mL were obtained for the limits of detection and quantification, respectively. The repeatability, inter-day precision, and recovery values ranges were 0.6%–4.5%, 5.4%–9.9%, and 83%–93%, respectively. Fresh and dried beans, as well as pods, cultivated exclusively with organic methods avoiding any synthetic fertilizers and pesticides were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight.     

An LC–MS/MS method for quantification of demethylzeylasteral,a novel immunosuppressive agent in rat plasma and the application to a pharmacokinetic study

In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of demethylzeylasteral in rat plasma. Electrospray ionization was operated in the negative ion mode while demethylzeylasteral and oleanolic acid (internal standard) were measured by selected reaction monitoring (demethylzeylasteral: m/z 479.2 → 436.0; oleanolic acid: m/z 454.9 → 407.2). This LC–MS/MS method had good selectivity, sensitivity, accuracy and precision. The pharmacokinetic profiles of demethylzeylasteral were subsequently examined in Wistar rats after oral or intravenous administration.     

A rapid LC‐MS/MS method for quantification of CSUOH0901, a novel antitumor agent,in rat plasma

CSUOH0901, a novel anticancer derivative of nimesulide, exhibits very promising anticancer activities in various cancer cell lines. In order to support further pharmacological and toxicological studies of this promising anticancer drug candidate, an LC‐MS/MS method was developed and validated in accordance with the US Food and Drug Administration guidelines. The drug molecules were extracted from plasma samples by protein precipitation and then analyzed with LC‐ESI‐MS/MS. An excellent analyte separation was achieved using a phenomenex C₁₈ column with a mobile phase of 90% methanol and 5 m m of ammonium formate. The validated linear dynamic range was between 0.5 and 100 ng/mL and the achieved correlation coefficient (r²) was >0.9996. The results of inter‐ and intra‐day precision and accuracy were satisfactory, that is, <12% for accuracy and within ±5% for precision at a low and high quality control concentrations, respectively. In addition, the analyte and internal standard (JCC76) were found to be stable under the storage conditions at ?20°C for about 2 months. Hence, the acquired results proved that the LC‐ESI‐MS/MS method developed is precise, accurate and selective for the quantification of CSUOH0901 in plasma, and can be used for pharmacokinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Study of tanshinone IIA tissue distribution in rat by liquid chromatography-tandem mass spectrometry method

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for determining tanshinone IIA in rat tissues. After a single step liquid-liquid extraction with diethyl ether, tanshinone IIA and loratadine (internal standard) was subjected to LC/MS/MS analysis using positive electro-spray ionization under selected reaction monitoring mode. Chromatographic separation of tanshinone IIA and loratadine was achieved on a Hypersil BDS C(18) column (i.d. 2.1 x 50 mm, 5 microm) with a mobile phase consisting of methanol-1% formic acid (90:10, v/v) at a flow rate of 300 microL/min. The intra-day and inter-day precision of the method were less than 10.2 and 12.4%, respectively. The intra-day and inter-day accuracies ranged from 99.7 to 109.7%. The lowest limit of quantification for tanshinone IIA was 1 ng/mL. The method was applied to a tanshinone IIA tissue distribution study after an oral dose of 60 mg/kg to rats. Tanshinone IIA tissue concentrations decreased in the order of stomach > small intestine > lung > liver > fat > muscle > kidneys > spleen > heart > plasma > brain > testes. Tanshinone IIA still could be detected in most of the tissues at 20 h post-dosing. These results indicate that the LC/MS/MS method was rapid and sensitive to quantify tanshinone IIA in different rat tissues.     

Development and validation of an LC-MS/MS method for profiling 39 urinary steroids (estrogens,androgens, corticoids,and progestins)

Abnormal production or metabolism of steroid hormones is responsible for the development of endocrine diseases. Thus, accurate quantification of steroid hormones is needed for both research into clinical conditions and diagnostic and monitoring purposes. An improved analytical method for profiling 39 steroids in urine using LC–MS/MS was developed. As a pre-treatment procedure prior to LC–tandem mass spectrometry (LC–MS/MS) analysis, hydrolysis using β-glucuronidase and solid-phase extraction for purifying the samples were performed. Steroids were separated using Waters ACQUITY BEH C₁₈ column (2.1 × 100 mm, 1.7 μm) and a mobile phase consisting of eluent A (0.01% formic acid and 1 mm ammonium formate in water) and eluent B (0.01% formic acid and 1 mm ammonium formate in methanol) with a gradient program at a flow rate of 0.4 mL/min. Under the optimized method, the linearity of calibration curves was higher than 0.992. The limits of detection at signal-to-noise ratio of 3 were 0.03–90 ng/mL. The developed novel LC–MS/MS method can quantitatively profile 39 steroids in a single analytical run. Steroid profiling based on quantitative results could improve the diagnosis and monitoring of hormone-dependent diseases.     

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19-nortestosterone

The lateral flow strip test for 19-nortestosterone is one kind of immunochromatographic assay. Nitrocellulose membrane was separately immobilized with goat anti-rabbit IgG (control line) and 19-NT-OVA conjugate (test line). Anti-19-NT polyclonal antibody labeled with colloidal gold particles acted as the detector reagent. The assay is qualitatively, not quantitatively, judged with positive or negative result. We tested the sensitivity of the strip using spiked swine urine, and each specimen was independently measured by LC/MS/MS. The sensitivity, measure by eye, was determined to be 200 ng/mL. The assay time was less than 15 min, and so suitable for on-site rapid test.     

Improvements in protein identification confidence and proteome coverage for human liver proteome study by coupling a parallel mass spectrometry/mass spectrometry analysis with multi-dimensional chromatography separation

Recently, matrix-assisted laser desorption ionization (MALDI) technique has been shown to be complementary to electrospray ionization (ESI) with respect to the population of peptides and proteins that can be detected. In this study, we tried to hyphenate MALDI-TOF-TOF-MS and ESI-QUADRUPOLE-TOF-MS with a single 2D liquid chromatography for complicated protein sample analysis. The effluents of RPLC were split into two parts for the parallel MS/MS detection. After optimizing the operation conditions in LC separation and MS identification, a total of 1149 proteins were identified from the global lysate of normal human liver (NHL) tissue. Compared to the single MS/MS detection, the combined analysis increased the number of proteins identified (more than 25%) and enhanced the protein identification confidence. Proteins identified were categorized and analyzed based upon their cellular location, biological process and molecular function. The identification results demonstrated the application potential of a parallel MS/MS analysis coupled with multi-dimensional LC separation for complicated protein sample identification, especially for proteome analysis, such as human tissues or cells extracts.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of cefuroxime lysine in rat brain microdialysates by ultra‐fast liquid chromatography with UV and tandem mass spectrometry: application to an acute toxicokinetic study

Cefuroxime lysine is a new second‐generation cephalosporins, which can penetrate the blood–brain barrier to cure the meningitis. In order to investigate its acute toxicokinetic study after intraperitoneal injection of 675 mg/kg cefuroxime lysine, a sensitive and clean ultra‐fast liquid chromatography–tandem mass spectrometry (UFLC‐MS/MS) method for the determination of cefuroxime lysine in microdialysate samples was developed and validated, which was compared with UFLC‐UV as a reference method. Chromatographic separation was performed on a Shim‐pack XR‐ODS C₁₈ column (75 × 3.0 mm, 2.2 µm), with an isocratic elution of 0.1% formic acid in acetonitrile–0.1% formic acid in water (45:55, v/v) for LC‐MS and acetonitrile–20 mm potassium dihydrogen phosphate (pH 3.0,20:80, v/v) for LC‐UV. The lower limit of detection was 0.01 µg/mL for LC‐MS and 0.1 µg/mL for LC‐UV method, with the same corresponding linearity range of 0.1–50 µg/mL. The intra‐ and inter‐day precisions (relative standard deviation) for both methods were from 1.1 to 8.9%, while the accuracy was all within ±10.9%. The results of both methods were finally compared using paired t‐test; the results indicated that the concentrations measured by the two methods correlated significantly (p < 0.05), which suggested that the two methods based on LC‐MS and LC‐UV were suitable for the acute toxicokinetic study. Copyright © 2014 John Wiley & Sons, Ltd.