Analytical validation and clinical application of urinary vanillylmandelic acid and homovanillic acid by LC–MS/MS for diagnosis of neuroblastoma

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinical biomarkers for diagnosis of neuroblastoma (NB), which commonly occurs in the childhood. Development and application of a robust LC–MS/MS method for fast determination of these biomarkers for optimal laboratory testing of NB is essential in clinical laboratories. In present study, we developed and validated a simple liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quick clinical testing of VMA and HVA for diagnosis of NB. The method was validated according to the current CLSI C62‐A and FDA guidelines. The age‐adjusted pediatric reference intervals and diagnostic performance were evaluated in both 24 h urine and random urine. Injection‐to‐injection time was 3.5 min. Inter‐ and intra‐assay coefficients of variation (CVs) were ≤3.88%. The lower limit of quantification and the limit of detection were 0.50 and 0.25 μmol/L for both VMA and HVA. Recoveries of VMA and HVA were in the ranges of 85–109% and 86–100% with CVs ≤5.76%. This method was free from significant matrix effect, carryover and interference. The establishment of age‐adjusted pediatric reference intervals by this LC–MS/MS method was favorable for the improvement in diagnostic performance, which was crucial for correct interpretation of test results from children in both 24 h and random urine.     

Determination of serum catecholamine metabolites in neuroblastoma by high performance liquid chromatography with electrochemical detection

A method for determining serum catecholamine metabolites such as vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenyl glycol (MHPG) and homovanillic acid (HVA) in neuroblastoma by using high performance liquid chromatography and electrochemical detector is described. The separation of catecholamine metabolites was performed on a reverse phase column with an eluting system containing citric acid-potassium hydrogen phosphate buffer and methanol as the organic modifier. The experimental results showed that VMA and HVA levels in the serum of neuroblastoma patients were 15-30 times higher than that of the normal control group. The same phenomenon also occurred in patients with stage II neuroblastoma. Serum VMA, MHPG and HVA levels reduced to normal in patients suffering from neuroblastoma after surgery. Serum catecholamine metabolites analysed by using HPLC/ECD is more simple, sensitive and reliable than that by usual urine assay and might be used for the diagnosis of neuroblastoma even in early stage.     

Separation and determination of homovanillic acid and vanillylmandelic acid by capillary electrophoresis with electrochemical detection

A method of separation and determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA) was developed based on capillary zone electrophoresis/amperometric detection with high sensitivity, good resolution and selectivity. In order to achieve complete separation and good response, several factors including pH, buffer concentration, separation voltage, detection potential and the length of separation capillary, were studied in detail. The method has been used to determine both HVA and VMA in human urine. Uric acid (UA) in human urine did not interference with their determination. The limit of detection of the method was 1.3×10⁻⁶ mol/l (1.4 fmol) for HVA and 7.9×10⁻⁷ mol/l (0.87 fmol) for VMA at a signal-to-noise ratio of 3.     

LC–MS/MS method for the simultaneous determination of tenofovir,emtricitabine, elvitegravir and rilpivirine in dried blood spots

A simple, short, and rugged LC–MS/MS method for the simultaneous determination of tenofovir, emtricitabine, elvitegravir and rilpivirine was developed and validated. Dried blood spots were prepared with 25 μL of spiked whole blood. A 3 mm punch was extracted with methanol containing labeled internal standards. Ten microliters was injected into the LC–MS/MS using isocratic mobile phase composed of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (45: 55 v/v) at a flow rate of 0.25 mL/min. The method was validated in the range of 10–2000 ng/mL for all four analytes. The intra‐assay accuracy (RE) of the method was −4.73–4.78, 1.35–2.89, −8.89 to −0.49 and − 1.40–1.81 for tenofovir, emtricitabine, elvitegravir and rilpivirine, respectively. The inter‐assay accuracy was within ±15% of nominal and precision (CV) was <15%. The hematocrit effect on quantification was nonsignificant at the tested hematocrit levels (35–70%). The dried blood spot method showed good agreement with the plasma method, and hence can be used as an alternative to plasma method.     

Online In-Tube Solid-Phase Microextraction Coupled to Liquid Chromatography–Tandem Mass Spectrometry for the Determination of Tobacco-Specific Nitrosamines in Hair Samples

Active and passive smoking are serious public health concerns Assessment of tobacco smoke exposure using effective biomarkers is needed. In this study, we developed a simultaneous determination method of five tobacco-specific nitrosamines (TSNAs) in hair by online in-tube solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC–MS/MS). TSNAs were extracted and concentrated on Supel-Q PLOT capillary by in-tube SPME and separated and detected within 5 min by LC–MS/MS using Capcell Pak C18 MGIII column and positive ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. The calibration curves of TSNAs showed good linearity in the range of 0.5–1000 pg mL^–1 using their stable isotope-labeled internal standards. Moreover, the limits of detection (S/N = 3) of TSNAs were in the range of 0.02–1.14 pg mL^–1, and intra-day and inter-day precisions were below 7.3% and 9.2% (n = 5), respectively. The developed method is highly sensitive and specific and can easily measure TSNA levels using 5 mg hair samples. This method was used to assess long-term exposure levels to tobacco smoke in smokers and non-smokers.     

Determination of three Tumor Biomarkers (Homovanillic Acid,Vanillylmandelic Acid,and 5‐Hydroxyindole‐3‐Acetic Acid) Using Flow Injection Analysis with Amperometric Detection

Flow injection analysis with amperometric detection (FIA‐AD) at screen‐printed carbon electrodes (SPCEs) in optimum medium of Britton‐Robinson buffer (0.04 mol ? L^?1, pH 2.0) was used for the determination of three tumor biomarkers (homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA)). Dependences of the peak current on the concentration of biomarkers were linear in the whole tested concentration range from 0.05 to 100 μmol ? L^?1, with limits of detection (LODs) of 0.065 μmol ? L^?1 for HVA, 0.053 μmol ? L^?1 for VMA, and 0.033 μmol ? L^?1 for 5‐HIAA (calculated from peak heights), and 0.024 μmol ? L^?1 for HVA, 0.020 μmol ? L^?1 for VMA, and 0.012 μmol ? L^?1 for 5‐HIAA (calculated from peak areas), respectively.     

HS–SPME–GC–MS and Electronic Nose Reveal Differences in the Volatile Profiles of Hedychium Flowers

Floral fragrance is one of the most important characteristics of ornamental plants and plays a pivotal role in plant lifespan such as pollinator attraction, pest repelling, and protection against abiotic and biotic stresses. However, the precise determination of floral fragrance is limited. In the present study, the floral volatile compounds of six Hedychium accessions exhibiting from faint to highly fragrant were comparatively analyzed via gas chromatography–mass spectrometry (GC–MS) and Electronic nose (E-nose). A total of 42 volatile compounds were identified through GC–MS analysis, including monoterpenoids (18 compounds), sesquiterpenoids (12), benzenoids/phenylpropanoids (8), fatty acid derivatives (2), and others (2). In Hedychium coronarium ‘ZS’, H. forrestii ‘Gaoling’, H. ‘Jin’, H. ‘Caixia’, and H. ‘Zhaoxia’, monoterpenoids were abundant, while sesquiterpenoids were found in large quantities in H. coccineum ‘KMH’. Hierarchical clustering analysis (HCA) divided the 42 volatile compounds into four different groups (I, II, III, IV), and Spearman correlation analysis showed these compounds to have different degrees of correlation. The E-nose was able to group the different accessions in the principal component analysis (PCA) corresponding to scent intensity. Furthermore, the pattern-recognition findings confirmed that the E-nose data validated the GC–MS results. The partial least squares (PLS) analysis between floral volatile compounds and sensors suggested that specific sensors were highly sensitive to terpenoids. In short, the E-nose is proficient in discriminating Hedychium accessions of different volatile profiles in both quantitative and qualitative aspects, offering an accurate and rapid reference technique for future applications.     

Screening of Potential Thrombin and Factor Xa Inhibitors from the Danshen–Chuanxiong Herbal Pair through a Spectrum–Effect Relationship Analysis

In this study; a spectrum–effect relationship analysis combined with a high-performance liquid chromatography–mass spectrometry (LC–MS) analysis was established to screen and identify active components that can inhibit thrombin and factor Xa (THR and FXa) in Salviae Miltiorrhizae Radix et Rhizoma–Chuanxiong Rhizoma (Danshen–Chuanxiong) herbal pair. Ten potential active compounds were predicted through a canonical correlation analysis (CCA), and eight of them were tentatively identified through an LC–MS analysis. Furthermore; the enzyme inhibitory activity of six available compounds; chlorogenic acid; Z-ligustilide; caffeic acid; ferulic acid; tanshinone I and tanshinone IIA; were tested to verify the feasibility of the method. Among them; chlorogenic acid was validated to possess a good THR inhibitory activity with IC₅₀ of 185.08 µM. Tanshinone I and tanshinone IIA are potential FXa inhibitors with IC₅₀ of 112.59 µM and 138.19 µM; respectively. Meanwhile; molecular docking results show that tanshinone I and tanshinone IIA; which both have binding energies of less than −7.0 kcal·mol⁻¹; can interact with FXa by forming H-bonds with residues of SER214; GLY219 and GLN192. In short; the THR and FXa inhibitors in the Danshen–Chuanxiong herbal pair have been successfully characterized through a spectrum–effect relationship analysis and an LC–MS analysis.     

A Validated LC–MS/MS Assay for the Simultaneous Quantification of the FDA-Approved Anticancer Mixture (Encorafenib and Binimetinib): Metabolic Stability Estimation

The concurrent use of oral encorafenib (Braftovi, ENF) and binimetinib (Mektovi, BNB) is a combination anticancer therapy approved by the United States Food and Drug Administration (USFDA) for patients with BRAFV600E/V600K mutations suffering from metastatic or unresectable melanoma. Metabolism is considered one of the main pathways of drug elimination from the body (responsible for elimination of about 75% of known drugs), it is important to understand and study drug metabolic stability. Metabolically unstable compounds are not good as they required repetitive dosages during therapy, while very stable drugs may result in increasing the risk of adverse drug reactions. Metabolic stability of compounds could be examined using in vitro or in silico experiments. First, in silico metabolic vulnerability for ENF and BNB was investigated using the StarDrop WhichP450 module to confirm the lability of the drugs under study to liver metabolism. Second, we established an LC–MS/MS method for the simultaneous quantification of ENF and BNB applied to metabolic stability assessment. Third, in silico toxicity assessment of ENF and BNB was performed using the StarDrop DEREK module. Chromatographic separation of ENF, BNB, and avitinib (an internal standard) was achieved using an isocratic mobile phase on a Hypersil BDS C₁₈ column. The linear range for ENF and BNB in the human liver microsome (HLM) matrix was 5–500 ng/mL (R² ≥ 0.999). The metabolic stabilities were calculated using intrinsic clearance and in vitro half-life. Furthermore, ENF and BNB did not significantly influence each other’s metabolic stability or metabolic disposition when used concurrently. These results indicate that ENF and BNB will slowly bioaccumulate after multiple doses.     

On the Identification and Quantification of Ergothioneine and Lovastatin in Various Mushroom Species: Assets and Challenges of Different Analytical Approaches

In recent years, mushrooms have drawn the attention of agro-industries and food-industries as they were considered to be valuable natural sources of health promoting compounds such as β-glucans, ergothioneine, and lovastatin. The detection and quantification of such compounds by implementing reliable analytical approaches is of the utmost importance in order to adjust mushrooms’ cultivation conditions and maximize the production in different species. Toward this direction, the current study focuses on the comparison of ultraviolet–visible (UV–Vis) spectrometry and liquid chromatography–mass spectrometry (LC–MS) methods (a) by evaluating the content of ergothioneine and lovastatin in mushrooms and (b) by highlighting any possible substrate-based interferences that hinder the accurate determination of these two compounds in order to propose the technique-of-choice for a standardized bioactive compounds monitoring. For this purpose, mushrooms produced by three species (i.e., Agaricus bisporus, Pleurotus ostreatus, and P. citrinopileatus) on various cultivation substrates, namely wheat straw (WS), winery (grape marc (GM)), and olive oil (OL) by-products, were examined. Among the two applied techniques, the developed and validated LC–MS methods, exhibiting relatively short analysis time and higher resolution, emerge as the methods-of-choice for detecting ergothioneine and lovastatin in mushrooms. On the contrary, UV–Vis methods were hindered due to co-absorbance of different constituents, resulting in invalid results. Among the studied mushrooms, P. citrinopileatus contained the highest amount of ergothioneine (822.1 ± 20.6 mg kg⁻¹ dry sample), whereas A. bisporus contained the highest amounts of lovastatin (1.39 ± 0.014 mg kg⁻¹ dry sample). Regarding the effect of different cultivation substrates, mushrooms produced on OL and WS contained the highest amount of ergothioneine, while mushrooms deriving from GM-based substrates contained the highest amount of lovastatin.     

Direct Flavonoid-Focused Chemical Comparison among Three Epimedium Plants by Online Liquid Extraction–High Performance Liquid Chromatography–Tandem Mass Spectrometry

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC–MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC–MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC–MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M–H]⁻). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn’t occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.     

Magnetic Solid-Phase Extraction Based on Magnetic Sulfonated Reduced Graphene Oxide for HPLC–MS/MS Analysis of Illegal Basic Dyes in Foods

In this study, a magnetic solid-phase extraction (MSPE) method coupled with High-Performance Liquid Chromatography Mass Spectrometry (HPLC–MS/MS) for the determination of illegal basic dyes in food samples was developed and validated. This method was based on Magnetic sulfonated reduced graphene oxide (M-S-RGO), which was sensitive and selective to analytes with structure of multiaromatic rings and negatively charged ions. Several factors affecting MSPE efficiency such as pH and adsorption time were optimized. Under the optimum conditions, the calibration curves exhibited good linearity, ranging from 5 to 60 µg/g with correlation coefficients >0.9950. The limits of detection of 16 basic dyes were in the range of 0.01–0.2 µg/L. The recoveries ranged from 70% to 110% with RSD% < 10%. The results indicate that M-S-RGO is an efficient and selective adsorbent for the extraction and cleanup of basic dyes. Due to the MSPE procedures, matrix effect and interference were eliminated in the analysis of HPLC–MS/MS without the matrix-matched standards. Thus, validation data showed that the proposed MSPE–HPLC–MS/MS method was rapid, efficient, selective, and sensitive for the determination of illegal basic dyes in foods.     

Improved liquid chromatography–tandem mass spectrometry method in clinical utility for the diagnosis of Cushing’s syndrome

Determination of urinary free cortisol is one of the first lines in screening for the diagnosis of Cushing’s syndrome where its measurement is mostly done by immunoassay. Although easy to perform, immunoassays suffer from the problem of assay interferences and are unable to measure cortisone levels. To enhance such techniques for clinical diagnosis, an improved liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the simultaneous determination of urinary free cortisol and cortisone. The leftover urine samples from immunoassay were collected and subjected to facile solid-phase extraction cleanup. In the analysis of 130 urine samples from patients, 65 (50%) were found to have elevated urinary free cortisol (UFC) by immunoassay; but only 13 (10.8%) were found to have elevated UFC by this improved LC–MS/MS method. Nine out of the 13 patients, which showed elevated UFC by LC–MS/MS, were surgically confirmed to have Cushing’s syndrome/disease. By setting a two times upper limit as a cut-off, the immunoassay gave a positive predictive value of 43.5%, whilst by using the improved method, a positive predictive value of 90% was obtained. Although several tests have been used extensively in first line screening for the diagnosis of Cushing’s syndrome, none has ever shown with full capability of distinguishing all cases of Cushing’s syndrome from normal and/or obese individuals. This method has shown superior analytical advantages over existing immunoassay type in terms of sensitivity, specificity and capability to diagnose Cushing’s syndrome. Comparison between existing spectrometric methods, the reported developed method shown here, provides a simpler sample preparation procedure and meets with the high throughput demand of clinical laboratories.     

Self-quenching in the electrochemiluminescence of tris(2,2'-bipyridyl) ruthenium(ii) using metabolites of catecholamines as co-reactants

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) using metabolites of catecholamines: homovanillic acid (HVA) and vanillylmandelic acid (VMA) as co-reactants were investigated in aqueous solution for the first time. When HVA and VMA were co-existent in the buffer solution containing Ru(bpy)(3)(2+), ECL peaks were observed at a potential corresponding to the oxidation of Ru(bpy)(3)(2+), and the ECL intensity was increased noticeably when the concentrations of HVA and VMA were at lower levels. The linear calibration range was from 8.0 × 10(-5) to 1.0 × 10(-9) M for HVA and VMA. The detection limit (S/N = 3) of HVA and VMA was 4.0 × 10(-10) M. The formation of the excited state Ru(bpy)(3)(2+*) was confirmed to result from the reaction between Ru(bpy)(3)(3+) and the intermediates of HVA or VMA radicals. Moreover, it was found that the ECL intensity was quenched significantly when the concentrations of HVA and VMA were relatively higher. The mechanism of self-quenching processes involved in the Ru(bpy)(3)(2+)-HVA and -VMA ECL systems are proposed in this study.     

Application of Multilayer Analytical Film to ELISA of Vanilmandelic Acid

The urinary level of vanilmandelic acid (VMA) is one marker for diagnosis of neurblastoma in neonates. A large number of samples are needed for mass screening. In order to make a convenient and sophisticated assay, we examined a method based on dry chemistry by using a monoclonal antibody to VMA. The antibody adsorbed on the surface of the well of a microplate was equilibrated with VMA labeled with glucose oxidase (GOD-VMA). Free VMA in a sample competes with GOD-VMA for the binding sites of the antibody. An aliquot containing the excess GOD-VMA was spotted onto a multilayer film consisting of a spreading layer containing glucose and a reaction layer containing peroxidase and a leucodye. Hydrogen peroxide produced with GOD-VMA in the spreading layer penetrated the reaction layer and produced a blue dye. The amount of dye formed was measured by a reflection photometer. From a standard curve, VMA was measurable from 0.1 to 50 μg/ml. The values obtained by the present method were correlated with those by high-performance liquid chromatography and enzyme-linked immunosorbent assay.     

Simultaneous Quantification of 25 Fentanyl Derivatives and Metabolites in Oral Fluid by Means of Microextraction on Packed Sorbent and LC–HRMS/MS Analysis

Fentanyl and fentalogs’ intake as drugs of abuse is experiencing a great increase in recent years. For this reason, there are more and more cases in which it is important to recognize and quantify these molecules and related metabolites in biological matrices. Oral fluid (OF) is often used to find out if a subject has recently used a psychoactive substance and if, therefore, the person is still under the effect of psychotropics. Given its difficulty in handling, good sample preparation and the development of instrumental methods for analysis are essential. In this work, an analytical method is proposed for the simultaneous determination of 25 analytes, including fentanyl, several derivatives and metabolites. OF was collected by means of passive drool; sample pretreatment was developed in order to be fast, simple and possibly semi-automated by exploiting microextraction on packed sorbent (MEPS). The analysis was performed by means of LC–HRMS/MS obtaining good identification and quantification of all the analytes in less than 10 min. The proposed method was fully validated according to the Scientific Working Group for Forensic Toxicology (SWGTOX) international guidelines. Good results were obtained in terms of recoveries, matrix effect and sensitivity, showing that this method could represent a useful tool in forensic toxicology. The presented method was successfully applied to the analysis of proficiency test samples.     

The validation of a simple LC/MS/MS method for determining the level of mevalonic acid in human plasma

A simple plasma extraction method coupled with liquid chromatography–tandem mass spectrometry (LC/MS/MS) detection was developed and validated for the analysis of endogenous mevalonic acid (MVA), a biomarker indicative of the rate of cholesterol biosynthesis, in human plasma samples. The analyte was extracted from the plasma matrix using a straightforward liquid–liquid sample preparation procedure. The extract supernatants were evaporated, reconstituted in aqueous solvent and injected into the LC/MS/MS system without further processing. The chromatographic separation was achieved on a reverse‐phase high‐performance liquid chromatography column. The accuracy and precision of the method was determined over the concentration range 0.25–25 ng/mL MVA from human plasma extracts in three validation batch runs. Inter‐assay precision (%CV) and accuracy (%RE) of the quality control samples were ≤7.00% (at lower limit quality control) and ≤6.10%, respectively. The sensitivity and throughput of this assay was significantly improved relative to previously published methods, resulting in smaller sample requirements and shorter analysis time. Assay results from a clinical study following the oral administration of an exploratory statin demonstrate that this procedure could potentially be used in the investigation of therapies associated with hypercholesterolemia. Copyright © 2010 John Wiley & Sons, Ltd.     

UPLC-MS/MS法测定高尿酸血症大鼠血清及尿液中的尿酸和肌酐

建立了一种测定血清和尿液中尿酸和肌酐含量的超高效液相色谱-串联质谱联用（UPLC-MS/MS）方法. 样品经自动进样器引入液相色谱通过预柱富集和去除杂质后,直接引入质谱中进行分析. 采用正离子电喷雾电离模式下的多反应监控模式对肌酐进行定量分析,纯溶剂标准曲线的肌酐线性范围为0.03~400 μmol/L;基质标准曲线的肌酐线性范围为0.2~400 μmol/L;它们的最低定量限分别为0.03和0.2 μmol/L. 采用负离子电喷雾电离模式下的多反应监控模式对尿酸进行定量分析,纯溶剂标准曲线的尿酸线性范围为0.1~350 μmol/L;基质标准曲线的尿酸线性范围为0.5~300 μmol/L;它们的最低定量限分别为0.1和0.5 μmol/L. 该方法的提取回收率在91.8%~103.7%之间,日内和日间RSD分别小于5.9%和6.8%,满足生物分析的要求. 利用该方法进行抗痛风中药筛选及其作用机制研究,结果表明,与阳性对照药物别嘌呤醇和苯溴马隆相比,中药二妙丸、黄柏和苍术均能在一定程度上降低血尿酸水平,并可显著逆转肾功能损伤,对肾功能具有一定的保护作用.     

N-Nitrosodimethylamine Contamination in the Metformin Finished Products

A GC–MS/MS method with EI ionization was developed and validated to detect and quantify N-nitrosodimethylamine (NDMA) and seven other nitrosamines in 105 samples of metformin tablets from 13 different manufactures. Good linearity for each compound was demonstrated over the calibration range of 0.5–9.5 ng/mL. The assay for all substances was accurate and precise. NDMA was not detected in the acquired active pharmaceutical ingredient (API); however, NDMA was detected in 64 (85.3%) and 22 (91.7%) of the finished product and prolonged finished product samples, respectively. European Medicines Agency recommends the maximum allowed limit of 0.032 ppm in the metformin products. Hence, 28 finished products and 7 pronged dosage products were found to exceed the acceptable limit of daily intake of NDMA contamination. The implications of our findings for the testing of pharmaceutical products are discussed.     

Development,Validation, and Comparison of Two Mass Spectrometry Methods (LC-MS/HRMS and LC-MS/MS) for the Quantification of Rituximab in Human Plasma

Rituximab is a chimeric immunoglobulin G1-kappa (IgG1κ) antibody targeting the CD20 antigen on B-lymphocytes. Its applications are various, such as for the treatment of chronic lymphoid leukemia or non-Hodgkin’s lymphoma in oncology, and it can also be used in the treatment of certain autoimmune diseases. Several studies support the interest in therapeutic drug monitoring to optimize dosing regimens of rituximab. Thus, two different laboratories have developed accurate and reproductive methods to quantify rituximab in human plasma: one using liquid chromatography quadripolar tandem mass spectrometer (LC-MS/MS) and the other, liquid chromatography orbitrap tandem mass spectrometer (LC-MS/HRMS). For both assays, quantification was based on albumin depletion or IgG-immunocapture, surrogate peptide analysis, and full-length stable isotope-labeled rituximab. With LC-MS/MS, the concentration range was from 5 to 500 µg/mL, the within- and between-run precisions were <8.5%, and the limit of quantitation was 5 µg/mL. With LC-MS/HRMS, the concentration range was from 10 to 200 µg/mL, the within- and between-run accuracy were <11.5%, and the limit of quantitation was 2 µg/mL. Rituximab plasma concentrations from 63 patients treated for vasculitis were compared. Bland–Altman analysis and Passing–Bablok regression showed the interchangeability between these two methods. Overall, these methods were robust and reliable and could be applied to routine clinical samples.