Semi-automated quantification of methylmalonic acid in human serum by LC-MS/MS

Abstract Background. Methylmalonic acid (MMA), a sensitive biomarker of functional vitamin B12 deficiency, is commonly determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using manual extraction and derivatization of MMA to reduce polarity prior to separation. Methods. In the present study we introduce a semi-automated extraction on a strong anion exchanger, HPLC separation on a BEH-amide column to separate serum MMA from its abundant isoform, succinic acid, followed by MS/MS detection and quantification. Results. The extraction of MMA plus internal standard provides full recovery and the method is linear between 0.03 μmol/L and 20.0 μmol/L (r(2) =?1.0) with intra-and inter-assay imprecision of 2.2%. Agreement with other laboratories has been demonstrated in external proficiency testing. Compared to both conventional GC-MS and LC-MS/MS methods, the correlation is r(2) >?0.99. Conclusions. The use of robotic pipetting, elimination of derivatization and improved separation by the BEH-amide column combined with HILIC chromatographic conditions significantly improve sample throughput compared to conventional methods. Using a single pipetting robot and LC-MS/MS instrument, this method is currently performing 180 analyses per day from10 regional hospitals and several additional distant sites.     

Investigation of the metabolic stability of olmutinib by validated LC-MS/MS: quantification in human plasma

Olmutinib (OTB, Olita™) is an orally available third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI). It was developed by Boehringer Ingelheim and Hanmi Pharmaceutical Co. Ltd for the cure of non-small cell lung cancer (NSCLC). In May 2016, OTB was approved in South Korea for the treatment of patients suffering from metastatic or locally advanced EGFR T790M mutation-positive NSCLC. A LC-MS/MS methodology was validated for OTB quantification in human plasma. An extended application for this validated LC-MS/MS is OTB metabolic stability evaluation. Chromatographic separation of OTB and ponatinib (PNT, IS) was attained using a reversed phase with isocratic elution. The linearity of the developed LC-MS/MS method ranged from 5.00 to 500.00 ng mL⁻¹ with r² ≥ 0.9999 in human plasma. LOD and LOQ were 1.12 and 3.39 ng mL⁻¹, respectively. The intra-day and inter-day precision and accuracy were 1.17 to 2.75% and 97.86 to 101.48%, respectively. The intrinsic clearance (CL_int) was 2.71 mL min⁻¹ kg⁻¹ and the in vitro half-life (t_1/2) was 48.80 min. A review of the literature revealed that there are no previous articles about the quantification of OTB in human plasma using LC-MS/MS or its metabolic stability assessment.

Olmutinib (OTB, Olita™) is an orally available third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI).     

Novel steroidal saponins with cytotoxic activities from the roots of Ophiopogon japonicus (L. f.) Ker-Gawl

Six new steroidal saponins (1–6) and one known steroidal saponin (7) were obtained from the roots of Ophiopogon japonicus (L. f.) Ker-Gawl. Their structures were determined by the detailed analysis of extensive nuclear magnetic resonance and mass spectroscopic data. The in vitro cytotoxic activities of these compounds against MDA-MB-435, HepG2 and A549 cell lines were also investigated.

Novel cytotoxic steroidal saponins from the roots of Ophiopogon japonicus (L. f.) Ker-Gawl.     

Current status and future developments of LC-MS/MS in clinical chemistry for quantification of biogenic amines

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is rapidly gaining ground in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review overviews current LC-MS/MS methods used for the quantification of biogenic amines and their metabolites. New possibilities offered by this technique are illustrated by recently developed assays for biogenic amines. Major shortcomings of conventional chromatographic techniques, such as labor-intensive sample preparation, long analysis times and often the relatively low specificity, are circumvented by using LC-MS/MS. In addition, LC-MS/MS has broad analyte compatibility and high analytical performance. In the last 5 years introduction of LC-MS/MS in routine diagnostics has resulted in improved assays for diagnosis and follow-up of neuroendocrine tumors characterized by the secretion of biogenic amines. Due to their labile nature and low concentration ranges biogenic amines require extensive and careful sample preparation. Introduction of new sophisticated techniques such as selective sorbents adsorption is evolving. This enables not only more specific analyte selection, but also automation of the complicated clean-up procedure. Automated sample clean-up can be directly coupled to LC-MS/MS, which facilitates reproducible and efficient handling of the growing number of samples to be analyzed in laboratories.     

LC-MS/MS quantification of Zn-alpha2 glycoprotein: a potential serum biomarker for prostate cancer

BACKGROUND: Zn-alpha2 glycoprotein (ZAG) is a relatively abundant glycoprotein that has potential as a biomarker for prostate cancer. We present a high-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring serum ZAG concentrations by proteolytic cleavage of the protein and quantification of a unique peptide. METHODS: We selected the ZAG tryptic peptide (147)EIPAWVPEDPAAQITK(162) as the intact protein for quantification and used a stable isotope-labeled synthetic peptide with this sequence as an internal standard. Standards using recombinant ZAG in bovine serum albumin, 50 g/L, and a pilot series of patient sera were denatured, reduced, alkylated, and digested with trypsin. The concentration of ZAG was calculated from a dose-response curve of the ratio of the relative abundance of the ZAG tryptic peptide to internal standard. RESULTS: The limit of detection for ZAG in serum was 0.08 mg/L, and the limit of quantification was 0.32 mg/L with a linear dynamic range of 0.32 to 10.2 mg/L. Replicate digests from pooled sera run during a period of 3 consecutive days showed intraassay imprecision (CV) of 5.0% to 6.3% and interassay imprecision of 4.4% to 5.9%. Mean (SD) ZAG was higher in 25 men with prostate cancer [7.59 (2.45) mg/L] than in 20 men with nonmalignant prostate disease [6.21 (1.65) mg/L, P = 0.037] and 6 healthy men [3.65 (0.71) mg/L, P = 0.0007]. CONCLUSIONS: This LC-MS/MS assay is reproducible and can be used to evaluate the clinical utility of ZAG as a cancer biomarker.     

Effect of six steroidal saponins isolated from anemarrhenae rhizoma on platelet aggregation and hemolysis in human blood.

Six steroidal saponins were isolated from Anemarrhena asphodeloides Bunge (Liliaceae), a traditional chinese medicine, and named anemarrhenasaponin I (An-I), anemarrhenasaponin Ia (An-Ia), timosaponin B-I (TB-I), timosaponin B-II (TB-II), timosaponin B-III (TB-III), and timosaponin A-III (TA-III). The effects of these six compounds on platelet aggregation and hemolysis in human blood were studied. All these compounds provoked remarkable inhibiting effect on platelet aggregation, and activated partial thromboplastin times (APTT) are sensitive to the presence of these six compounds. Using an in vitro system, APTT was delayed with the increment of the concentrations of these six compounds. In these six compounds, only timosaponin A-III appeared a strong effect on hemolysis, and anemarrhenasaponin Ia had a slight effect on hemolysis, other had no effect on hemolysis. These results suggested that these steroidal saponins isolated from Anemarrhena asphodeloides Bunge (Liliaceae) might be used as a novel antithrombotic therapeutic agents in post-myocardial infarction.     

串联质谱和高效液相色谱-串联质谱二次筛查联合应用于新生儿MMA筛查

目的探讨串联质谱和高效液相色谱-串联质谱二次筛查联合应用在甲基丙二酸血症(MMA)中的筛查价值。方法收集新生儿串联质谱初筛结果中C3、C3/C2、C3/C0单一或多个指标异常的新生儿干血滤纸片标本,用高效液相色谱-串联质谱的方法定量检测原始血片中甲基丙二酸、甲基枸橼酸和高半胱氨酸,对二次筛查后疑似阳性的新生儿进行召回复查,并进行尿气相色谱/质谱检测。临床诊断患儿进一步予以基因检测进行确诊。结果共收集423例C3、C3/C2、C3/C0单一或多个指标异常的新生儿筛查标本,初筛阳性率约为1%,行联合筛查检测结果发现8例标本中甲基丙二酸和同型半胱氨酸表达水平明显升高,召回复查尿气相色谱质谱提示甲基丙二酸轻度升高。结论串联质谱和高效液相色谱-串联质谱联合应用可以提高新生儿MMA筛查的阳性预测值、降低假阳性率,在新生儿遗传代谢病筛查中具有重要价值。     

Experience with therapeutic drug monitoring of three antifungal agents using an LC-MS/MS method in routine clinical practice

Therapeutic drug monitoring (TDM) of voriconazole, itraconazole, and posaconazole is a useful tool for treatment of fungal infections. We validated a simple and reliable LC-MS/MS method using simple protein precipitation for simultaneous determination of voriconazole, itraconazole, and posaconazole. The linearity, accuracy, precision, carryover, and matrix effects were validated. Total sample preparation time was <30 min per batch, and analytical run time was 3.8 min per sample. We also presented clinical experience of TDM at 1183 serum concentrations over one year using this validated assay method. About 77%, 85%, and 96% of measured voriconazole, itraconazole, and posaconazole concentrations were within the therapeutic range, respectively. The number of respective measurements per patient was 1–63, 1–8, and 1–4 for voriconazole, itraconazole, and posaconazole. All three antifungal agents showed large intra-individual variability (2 to 181% CV) and drug-drug interaction with proton pump inhibitors or rifampin. In conclusion, we developed and validated a simple and fast method that was successfully applied in a routine clinical setting.     

A validated LC-MS/MS analytical method for the quantification of pemigatinib: metabolic stability evaluation in human liver microsomes

Pemigatinib (PMB) is a small molecule inhibitor of fibroblast growth factor receptor 1 (FGFR1), FGFR2 and FGFR3. On April 17, 2020, the US Food and Drug Administration granted accelerated approval for PMB for the treatment of adults with previously treated, unresectable metastatic or locally advanced cholangiocarcinoma with a fibroblast growth factor receptor 2 (FGFR2) fusion or other rearrangement. PMB is considered the first targeted treatment for cholangiocarcinoma approved in the US. In this study, in silico prediction of PMB metabolic stability was done using the WhichP450 module of the StarDrop software package. Further, an LC-MS/MS analytical method was developed for PMB quantification in human liver microsomes (HLM) to experimentally assess metabolic stability. PMB and flavopiridol (FVL), used as an internal standard IS, were resolved using an isocratic mobile phase and a C18 stationary phase. The LC-MS/MS method showed linearity in the range of 5 to 500 ng mL⁻¹ in an HLM matrix (R² = 0.9995). The lower limit of quantification (LLOQ) was 5 ng mL⁻¹, indicating sensitivity. The inter- and intra-day accuracy and precision were within a variability of 10, confirming the reproducibility of the method. The measured in vitro half-life and intrinsic clearance of PMB were 27.29 min and 25.40 μL min⁻¹ mg⁻¹, respectively. PMB showed a moderate extraction ratio suggesting good bioavailability. The developed analytical method is the first LC-MS/MS method specific for PMB quantification with application to metabolic stability assessment.

PMB showed a moderate extraction ratio suggesting good bioavailability. The developed analytical method is the first LC-MS/MS method specific for PMB quantification with application to metabolic stability assessment.     

LC-MS/MS systematic toxicological analysis: comparison of MS/MS spectra obtained with different instruments and settings

OBJECTIVES: To compare the mass spectra obtained using a linear-ion-trap (LIT) tandem mass spectrometer (QTRAP) operated in the "enhanced product ion scan" (EPI) mode with those obtained in the classical triple-quadrupole product ion scan (PIS) mode run on the same as well as on two other instruments (TSQ-Quantum and Quattro-Micro). DESIGN AND METHODS: After tentative standardization of ion fragmentation and transmission in both polarities using a reference compound (glafenine) on the three instruments, eight test compounds detected in the positive mode and five in the negative mode were systematically infused in different ionization sources and spectral acquisition performed over approximately 5 s. The relative intensity of the ions present in the resulting spectra was quantitatively and statistically compared. Also, the intra-day and inter-day variabilities of these relative intensities, as well as the effect of increasing compound concentration, were studied using QTRAP operated in EPI mode. RESULTS: The EPI and PIS modes operated on a single LIT MS/MS instrument resulted in significant differences in relative ion intensities in both polarities, and so did the other two instruments despite prior standardization with glafenine. Some fragments could be absent in certain spectra, but no unexpected or unique fragments showed up. Intra-day variability was smaller in the LIT EPI than in the regular PIS mode and in the positive than in the negative polarity. In EPI mode, both intra- and inter-day variabilities increased when the relative intensity decreased. The effect of increasing concentration on the relative intensity of major and minor ions was small but significant in both polarities. Finally, contamination and cleansing of the ionization source also had noticeable effects on MS/MS spectra, though the cause is unclear. CONCLUSIONS: MS/MS spectra do not offer the expected inter-instrument reproducibility despite an attempt at standardizing the fragmentation conditions using a reference compound. However, although the inter-instrument differences in ion relative intensity were significant, the spectra obtained looked almost similar. This suggests that in library searching algorithms, higher weight should be assigned for the m/z ratios than for their relative intensity in the spectra.     

Enrichment and separation of steroidal saponins from the fibrous roots of Ophiopogon japonicus using macroporous adsorption resins

In this study, a simple and effective strategy for the enrichment of total steroidal saponins (TSS) from the fibrous roots of Ophiopogon japonicus (L. f.) Ker-Gawl. (FROJ) using macroporous adsorption resin was systematically developed. XAD-7HP resin was selected from six macroporous resins for further study because of the highest static adsorption and desorption capacities. The static adsorption of TSS on XAD-7HP resin fitted well to the Langmuir isotherm model and pseudo second-order kinetic model; the thermodynamics test showed that the adsorption process was spontaneous and exothermic. The dynamic tests on XAD-7HP resin columns demonstrated that the breakthrough volume was 16 bed volume (BV), and 6 BV of 80% ethanol was suitable for dynamic desorption. In a lab scale-up separation under optimal dynamic conditions, the content of TSS in the resin-enrichment fraction increased from 1.83% in the crude extracts to 13.86% by 7.59-fold with a recovery yield of 82.68%. Three steroidal saponins were obtained from the resin-enrichment fraction, and showed protective effects against oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cell (HUVEC) injury. Overall, these results suggested that XAD-7HP resin chromatography was an effective strategy for the large scale enrichment of TSS from FROJ, which showed the potential for functional food and pharmaceutical application.

In this study, a simple and effective strategy for the enrichment of total steroidal saponins (TSS) from the fibrous roots of Ophiopogon japonicus (L. f.) Ker-Gawl. (FROJ) using macroporous adsorption resin was systematically developed.     

Simultaneous quantification of vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in human serum by LC-MS/MS

Abstract

Introduction. Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D. Method. An LC-MS/MS method was developed and validated to quantify vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃ in the eluate were derivatized by 4-phenyl-1,2,4-triazoline-3,5-dione to improve sensitivity in the following LC-MS/MS analysis. Results. Using only 100 μL serum the limit of quantification was < 0.2 ng/mL for vitamin D₃, 25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D₃, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D₃ and up to 200 ng/mL (499 nmol/L) for 25-hydroxyvitamin D₃. Precision was < 6.5% for vitamin D₃ and 25-hydroxyvitamin D₃ and < 10.2% for 24,25-dihydroxyvitamin D₃. Conclusion. We demonstrate that a method including not only serum 25-hydroxyvitamin D₃ but also vitamin D₃ and 24,25-dihydroxyvitamin D₃ could easily be implemented in most modern biochemical laboratories. The method could be used to study the metabolism of endogenous synthesized vitamin D₃ as well as vitamin D₃ in intervention studies.     

Measurement of glycated albumin in serum and plasma by LC-MS/MS

Background Diagnosis of diabetes and monitoring of long-term blood sugar are preferably done by measurement of glycated hemoglobin (HbA1c). Diabetic patients with end stage renal disease (ESRD) may have short-lived red blood cells due to hemodialysis (HD), and thus higher turnover of hemoglobin. The level of glycated hemoglobin (HbA1c) may be lower than expected for these patients, even at increased blood glucose, possibly making glycated albumin (GA) measurement a better alternative. Methods The percentage of GA was measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fast and efficient trypsin digestion of proteins in diluted serum or plasma resulted in a high number of proteotypic peptides from albumin, including KQTALVELVK which was detected both glycated and non-glycated by multiple reaction monitoring (MRM). The percentage of GA was estimated by neat peak area response of glycated peptide divided by the sum of glycated and non-glycated peptide. Results Acceptable method reproducibility (6% CV), repeatability (2–6% CV), limit of quantification (0.75% GA), linearity (R²?=?0.999) and recovery (79?±?9%) was achieved without using calibration or isotope-labeled internal standard. GA was strongly correlated with HbA1c (r?=?0.84) for patients without ESRD. The average ratio of GA/HbA1c was significantly higher (p?=?0.0021) for ESRD patients (1.84?±?0.38, n?=?62) compared to other patients (1.67?±?0.28, n?=?225). Conclusion GA measurement by detecting glycation in KQTALVELVK with LC-MS/MS seems to be a useful supplement to HbA1c for detecting increased blood glucose in diabetic patients with ESRD.     

噁唑烷酮类新药MRX-Ⅰ在人血浆、尿液浓度超高效液相色谱串联质谱测定方法的建立及验证

目的建立及验证超高效液相色谱串联质谱（UPLC-MS,/MS）的方法用于测定人血浆和尿液中恶唑烷酮类新药MRX-I药物浓度。方法 UPLC-MS/MS液相条件为色谱柱Waters ACQUITY UPLC BEH C8;流动相为乙腈：水（40：60,v/v）。质谱采用ESI源正离子多反应监测（MRM）。内标为利奈唑胺,以乙酸乙酯液-液萃取法清除血浆及尿液样本中杂质。方法学验证包括基质效应、绝对回收率、精密度和准确度及MRX-I在人血浆及尿液样本中放置稳定性。结果 UPLC-MS/MS法检测MRX-I在人血浆和尿液中的线性范围均为（0.005 00～1.00）mg/L,最低检测浓度均为0.005 00 mg/L。MRX-I与内标在血浆和尿液中的保留时间小于1.5 min。本方法学验证结果显示MRX-I在人血浆和尿液基质效应因子分别为90.4%±8.2%和82.7%±7.9%;血浆和尿液中MRX-I提取回收率分别为112.8%±13.4%和105.6%±13.4%。MRX-I血浆样本的测定方法日内、日间准确度分别为98.9%～105.0%和96.5%～102.6%;尿液样本的测定方法日内、日间准确度分别为92.7%～98.6%和95.1%～105.7%。MRX-I在人血浆和尿液样本室温放置24 h、预处理后自动进样器放置48 h、-40℃冰箱冻融3次、-40℃冰箱分别放置8个月和6个月仍然保持稳定。结论本研究建立的UPLC-MS/MS检测人血浆及尿液中MRX-I浓度方法的灵敏度高,专属性强。其方法学验证结果均符合生物样品分析的要求。     

An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A,tacrolimus, sirolimus and everolimus in human whole blood

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and ¹³C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).     

重楼总皂苷对人肺癌细胞A549的增殖抑制作用及对细胞周期的影响

目的:研究重楼总皂苷对人肺癌细胞A549的增殖抑制作用及对细胞周期的影响.方法:用MTT法测定重楼总皂苷对A549细胞的增殖抑制率;Hoechst-PI双染观察细胞形态学变化;流式细胞术测定重楼总皂苷对A549细胞周期时相分布的影响.结果:重楼总皂苷能抑制A549细胞的增殖,其作用呈明显的时间和剂量依赖性;Hoechst-PI双染显示细胞凋亡特征;重楼总皂苷使A549细胞被阻滞在S期.结论:重楼总皂苷对A549细胞的增殖有较强的抑制作用,明显影响细胞周期时相分布.     

LC-MS/MS quantitation of ribavirin in serum and identification of endogenous isobaric interferences

BackgroundRibavirin is a nucleoside analog used in treatment of chronic hepatitis C. It is associated with severe, dose-dependent toxicities, including hemolytic anemia. To facilitate therapeutic drug monitoring, a liquid chromatography–tandem mass spectrometry method was validated for quantitation of ribavirin in serum.MethodsAfter protein precipitation, ribavirin is quantitated using a ¹³C₅-ribavirin internal standard, on a Hypercarb analytical column designed for retention of polar analytes.ResultsThe analytical method shows excellent precision, sensitivity, and specificity. In vitro drug stability was also assessed. Interestingly, endogenous isobaric compounds were noted in both human and bovine serum; these could be chromatographically separated from the ribavirin peak. Addition of exogenous uridine and cytosine increases the size of the isobaric peaks, suggesting that these compounds are the source of the endogenous interference.ConclusionsThis method uses mass spectrometric transitions that have been used in other published methods, but also separates ribavirin from isobaric peaks that were not described. These peaks were determined to be endogenous nucleosides. Laboratories quantitating ribavirin in biological matrices should be aware of the potential for isobaric interferences, and take steps to chromatographically separate them from the ribavirin peak for accurate quantitation.