Simultaneous chiral separation of ephedrine alkaloids by MEKC-ESI-MS using polymeric surfactant II: application in dietary supplements

Chiral MEKC-MS method was utilized for separation, identification, and quantitation of ten enantiomers of ephedrine and related compounds. Enantioselective separations of all ephedrine alkaloids were accomplished through a combination of polysodium N-undecenoxycarbonyl-L-leucinate (poly-L-SUCL) with 30% v/v ACN. Interestingly, the more hydrophilic stereoisomers were eluted later than the hydrophobic ones indicating that hydrogen bonding interactions are much stronger than hydrophobic interactions in the presence of ACN in chiral MEKC. The method was validated in terms of linearity, LOD, LOQ, precision and robustness. The method was finally used in the analysis of three standard reference materials (SRMs). Results of (-)-ephedrine ranged from 12.49 to 0.24 mg/g, for (+)-pseudoephedrine from 4.04 to 0.019 mg/g, for (-)-norephedrine from 0.36 to 0.0031 mg/g, for (+)-norpseudoephedrine from 0.68 to 0.0052 mg/g, for (-)-methylephedrine from 1.18 to 0.0092 mg/g and for (+)-methylpseudoephedrine from 0.086 to 0.00037 mg/g in the SRMs.     

Preparation and characterization of a suite of ephedra-containing standard reference materials

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.     

Enhanced sensitivity and resolution for the analysis of paralytic shellfish poisoning toxins in water using capillary electrophoresis with amperometric detection and field‐amplified sample injection

The profiling of the most lethal paralytic shellfish poisoning toxins (PSTs) in freshwater has increased the need to establish an alternative analytical method with high sensitivity and resolution. In this paper, a coupling technique of field‐amplified sample injection (FASI) and CE with end‐column amperometric detection (CE‐AD) was developed to improve the detection sensitivity and separation of PSTs by electrokinetically injecting a water plug of analytes to the capillary filled with a high‐conductivity BGE. Parameters affecting FASI and CE process were carefully adjusted to achieve the highest response and resolution. Separation selectivity for PSTs, especially for the analogues and epimers, was greatly enhanced by using 40 mM Britton–Robinson buffer (pH 9.5) as BGE, which altered the EOF and mobility of the analytes that interacted with polyborate ions. Satisfactory linear relationship between peak current and concentration of toxins were gained over a wide range of 1.95–254 μg/L. The detection limits (S/N = 3) for five PSTs ranged from 0.63 to 3.11 μg/L, which are below the health alert level in drinking water. In comparison with the up‐to‐date reporting chromatographic methods, the FASI‐CE‐AD method was simple, low‐cost, selective, and sensitive enough for direct quantification of PSTs at very low levels, implying a potential for screening and monitoring of PSTs in surface waters.     

Certification of standard reference materials containing bitter orange

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Definitive method certification of clinical analytes in lyophilized human serum: NIST Standard Reference Material (SRM) 909b

The National Institute of Standards and Technology (NIST) has developed several Standard Reference Materials (SRMs) based on human serum. NIST SRM 909b, Human Serum, is a lyophilized human serum material with concentrations for seven organic and six inorganic analytes at two levels certified solely by definitive methods (DMs). This material provides the vehicle by which high precision, high accuracy measurements made with DMs at NIST can be transferred through the measurement hierarchy to other laboratories. Isotope dilution gas chromatographic-mass spectrometric (GC-IDMS) methods were applied to measure cholesterol, creatinine, glucose, urea, uric acid, triglycerides, and total glycerides. Thermal ionization isotope dilution mass spectrometry (TI-IDMS) was used for determination of lithium, magnesium, potassium, calcium, and chloride. In addition, chloride was determined by coulometry, providing a comparison between two DMs. Sodium, which lacks a stable isotope that would permit isotope dilution mass spectrometric (IDMS) measurement, was determined by gravimetry. SRM 909b includes certified values for total glycerides and triglycerides, which were not certified in the previous lot of this material (SRM 909a). Improvement in uniformity of vial fill weight in the production of SRM 909b resulted in smaller certified uncertainties over previous freeze-dried serum SRMs. Uncertainties at the 99% level of confidence for relative expanded uncertainty (%) for certification of the organic analytes on a mmol/L/g basis ranged from 0.44% for urea (level II) to 5.04% for glucose (level II). (In-house studies have shown glucose to be a relatively unstable analyte in similar lyophilized serum materials, degrading at about 1% per year.) Relative expanded uncertainties (99% C.I.) for certification of inorganic analytes on a mmol/L/g basis ranged from 0.25% for chloride (level I) to 0.49% for magnesium (level II). Received: 30 July 1997 / Revised: 24 October 1997 / Accepted: 31 October 1997     

毛细管电泳结合压力辅助电动进样测定水样中4种酚类雌激素

在毛细管电泳的胶束电动色谱（MEKC）模式下,采用压力辅助电动进样（PAEKI）的进样方式在线富集4种酚类雌激素（PEs）。对影响PAEKI的进样电压、进样时间等进行考察,并与传统的压力进样比较。结果表明,在最优的PAEKI条件下（-9 kV,0.3 psi（约2.1 kPa）,0.4 min）,4种PEs在7 min内基线分离,线性关系良好,相关系数（r）大于0.9936,己烷雌酚和双烯雌酚的线性范围为0.05~5 mg/L、双酚A和己烯雌酚的线性范围为0.1~10 mg/L;检出限（S/N=3）为0.0071~0.017 mg/L,富集倍数为11~15。使用该MEKC-PAEKI法对自来水和湖水水样进行测定,得到定量限（S/N=10）分别为0.029~0.064 mg/L和0.033~0.079 mg/L;加标回收率为75.6%~110.1%,相对标准偏差（n=5）为4.6%~11.8%。PAEKI不需要使用其他试剂,只需对电泳仪的参数进行适当调整即可实现对分析物的在线富集,简单、快速、自动化程度高。     

Detection and validated quantification of nine herbal phenalkylamines and methcathinone in human blood plasma by LC-MS/MS with electrospray ionization

The herbal stimulants Ephedra species, Catha edulis (khat), and Lophophora williamsii (peyote) have been abused for a long time. In recent years, the herbal drug market has grown owing to publicity on the Internet. Some ingredients of these plants are also ingredients of cold remedies. The aim of the presented study is to develop a multianalyte procedure for detection and validated quantification of the phenalkylamines ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine, cathinone, mescaline, synephrine (oxedrine), and methcathinone in plasma. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a strong cation exchange separation column and gradient elution. They were detected using a Q-Trap LC-ESI-MS/MS system (MRM mode). Calibration curves were used for quantification using norephedrine-d3, ephedrine-d3, and mescaline-d9 as internal standards. The method was validated according to international guidelines. The assay was selective for the tested compounds. It was linear from 10 to 1000 ng/ml for all analytes. The recoveries were generally higher than 70%. Accuracy ranged from - 0.8 to 20.0%, repeatability from 2.5 to 12.3%, and intermediate precision from 4.6 to 20.0%. The lower limit of quantification was 10 ng/ml for all analytes. No instability was observed after repeated freezing and thawing or in processed samples. The applicability of the assay was tested by analysis of authentic plasma samples after ingestion of different cold medications containing ephedrine or pseudoephedrine, and after ingestion of an aqueous extract of Herba Ephedra. After ingestion of the cold medications, only the corresponding single alkaloids were detected in human plasma, whereas after ingestion of the herb extract, all six ephedrines contained in the plant were detected. The presented LC-MS/MS assay was found applicable for sensitive detection and accurate and precise quantification of all studied analytes in plasma.     

Definitive method certification of clinical analytes in lyophilized human serum: NIST Standard Reference Material (SRM) 909b

The National Institute of Standards and Technology (NIST) has developed several Standard Reference Materials (SRMs) based on human serum. NIST SRM 909b, Human Serum, is a lyophilized human serum material with concentrations for seven organic and six inorganic analytes at two levels certified solely by definitive methods (DMs). This material provides the vehicle by which high precision, high accuracy measurements made with DMs at NIST can be transferred through the measurement hierarchy to other laboratories. Isotope dilution gas chromatographic-mass spectrometric (GC-IDMS) methods were applied to measure cholesterol, creatinine, glucose, urea, uric acid, triglycerides, and total glycerides. Thermal ionization isotope dilution mass spectrometry (TI-IDMS) was used for determination of lithium, magnesium, potassium, calcium, and chloride. In addition, chloride was determined by coulometry, providing a comparison between two DMs. Sodium, which lacks a stable isotope that would permit isotope dilution mass spectrometric (IDMS) measurement, was determined by gravimetry. SRM 909b includes certified values for total glycerides and triglycerides, which were not certified in the previous lot of this material (SRM 909a). Improvement in uniformity of vial fill weight in the production of SRM 909b resulted in smaller certified uncertainties over previous freeze-dried serum SRMs. Uncertainties at the 99% level of confidence for relative expanded uncertainty (%) for certification of the organic analytes on a mmol/L/g basis ranged from 0.44% for urea (level II) to 5.04% for glucose (level II). (In-house studies have shown glucose to be a relatively unstable analyte in similar lyophilized serum materials, degrading at about 1% per year.) Relative expanded uncertainties (99% C.I.) for certification of inorganic analytes on a mmol/L/g basis ranged from 0.25% for chloride (level I) to 0.49% for magnesium (level II).     

Rapid selective accelerated solvent extraction and simultaneous determination of herbicide atrazine and its metabolites in fruit by ultra high performance liquid chromatography

A selective accelerated solvent extraction procedure achieved one step extraction and cleanup for analysis of herbicide atrazine and its metabolites in fruit. Using a BEH C18 analytical column and the gradient mode with 2 mM ammonium acetate aqueous solution/acetonitrile as a mobile phase achieved effective chromatographic separation of the five analytes within 4 min. The calibration curves were linear over two orders of magnitude of concentration with correlation coefficients (r) of 0.9996?0.9999. The method limit of quantification was 1, 2, 1.5, 3, and 2 μg/kg for atrazine, desethylatrazine, desisopropylatrazine, desethyldesisopropylatrazine, and hydroxyatrazine, respectively, in the case of atrazine it is at least two orders of magnitude lower than the maximum residue limit (0.25 mg/kg). The intra‐day and inter‐day precisions of the five analytes were in the range of 2.1–3.5 and 3.1–4.8 %, respectively. The recoveries of the five analytes at three spiked levels varied from 85.9 to 107% with a relative standard deviation of 1.8–4.9% for pear and apple samples. The ultra high performance liquid chromatography with diode array detection method was proved to be fast, inexpensive, selective, sensitive, and accurate for the quantification of the analytes in pear and apple samples.     

Thirty years of capillary electrophoresis in food analysis laboratories: potential applications

CE has generated considerable interest in the research community since instruments were introduced by different trading companies in the 1990s. Nowadays, CE is popular due to its simplicity, speed, highly efficient separations and minimal solvent and reagent consumption; it can also be included as a useful technique in the nanotechnology field and it covers a wide range of specific applications in different fields (chemical, pharmaceutical, genetic, clinical, food and environmental). CE has been very well evaluated in research laboratories for several years, and different new approaches to improve sensitivity (one of the main drawbacks of CE) and robustness have been proposed. However, this technique is still not well accepted in routine laboratories for food analysis. Researching in data bases, it is easy to find several electrophoretic methods to determine different groups of analytes and sometimes they are compared in terms of sensitivity, selectivity, precision and applicability with other separation techniques. Although these papers frequently prove the potential of this methodology in spiked samples, it is not common to find a discussion of the well-known complexity of the matrices to extract analytes from the sample and/or to study the interferences in the target analytes. Summarizing, the majority of CE scientific papers focus primarily on the effects upon the separation of the analytes while ignoring their behavior if these analytes are presented in real samples.     

A validated HPLC‐PDA method for identification and quantification of two bioactive alkaloids,ephedrine and cryptolepine,in different Sida species

A simple, rapid, accurate and reproducible reverse‐phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP‐18 X‐terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10–250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra‐day and inter‐day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.     

Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of acidic and neutral drugs

A multi‐analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on‐line solid‐phase extraction (SPE)–HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro‐neutral, one strong anion‐exchange, one weak cation‐exchange) for on‐line extraction, five HPLC‐columns [one C₁₈ (GeminiNX), two phenyl‐hexyl (Gemini C₆‐Phenyl, Kinetex Phenyl‐Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre‐treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion‐exchanger SPE cartridge (StrataX‐A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C₆‐Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile–water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter‐/intra‐day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from ?14 to 10%. A computer‐assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi‐drug intoxications are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Certification strategies for health-related SRMS at NIST

Inaccuracy in health-related measurements raises overall health care costs, results in misdiagnoses, leads to inaccurate conclusions in clinical studies, and results in inaccurate nutrition labeling. NIST has an extensive program aimed at providing the health measurements community with standard reference materials (SRMs) to assist them in making accurate measurements. A variety of approaches are used to certify health-related SRMs. For pure crystalline SRMs used as primary standards, direct assays of purity are often not possible. Thus numerous techniques including differential scanning calorimetry, mass spectrometry, chromatography, and others may be used to assess purity. For matrix SRMs used to assess the accuracy of measurement systems, the approaches for certification depend upon the needs of the users and NIST capabilities. When accuracy needs are the highest and the methods exist, NIST uses definitive methods, primarily involving isotope dilution mass spectrometry. These methods have been applied to the certification of serum-based SRMs for a number of the common clinical analytes. For many analytes, definitive methods have not been developed, so NIST uses other strategies for certification. In some cases, such as for drugs of abuse, two independent methods are used for the measurements. For nutrients such as vitamins, in-house methods are used along with results from outside laboratories having extensive experience with a particular analysis. The paper includes tables with examples of many of the health-related SRMs that are available. Received: 15 May 1997 / Revised: 23 July 1997 / Accepted: 25 July 1997     

毛细管电泳电化学检测法测定红葡萄酒中的多元酚

目前 ,人类各种疾病约 89%起因于活性氧 .因此消除活性氧基团 ,使过氧化物对机体的损伤降到最低限度已成为研究的热点 [1] . Maxwell等 [2 ] 测试了红葡萄酒在人体血液中的抗氧化能力 .发现从刚喝下红葡萄酒时起 ,抗氧化活性就开始上升 ,90 min后达到最大 ,抗氧化活性平均上升约 1 5 % .葡萄酒中的多酚含量与活性氧消除能力的相关系数高达 0 .9686,所以确立葡萄酒中多元酚的分析方法有重要意义 .红葡萄酒中含有酚酸类、儿茶素类、黄酮类等多酚类化合物 ,通常采用气相色谱[3] 、高效液相色谱 [4 ] 测定 .毛细管电泳 ( CE)应用于葡萄酒中多…     

快速高效液相色谱-串联质谱法同时测定水产品中5类33种药物残留

建立了快速高效液相色谱-串联质谱法(RRLC-MS/MS)同时测定水产品中四环素类、喹诺酮类、磺胺类、磺胺增效剂和三苯甲烷类共5大类33种药物残留的方法。样品用Na2EDTA-Mcllvaine缓冲溶液及乙腈提取,正己烷脱脂后,用RRLC进行分离。在电喷雾正离子模式下,以动态多反应监测(Dynamic MRM)方式采集数据进行定性与定量分析。33种药物在相应的浓度范围内线性良好,相关系数均大于0.99;在3个不同浓度添加水平下,平均回收率为63.6%～115.2%;相对标准偏差(RSD)为4.6%～14.6%;检出限(LOD,S/N=3)和定量限(LOQ,S/N=10)分别为0.1～2.0μg/kg和0.5～5.0μg/kg。本方法简便快速、灵敏可靠,适用于水产品中药物多残留的同时快速定性与定量测定。     

On-line pervaporation-capillary electrophoresis for the determination of volatile acidity and free sulfur dioxide in wines

Pervaporation has been coupled on-line to capillary electrophoresis (CE) by a simple interface consisting of a modified CE vial. The approach allows volatile analytes to be removed and injected into the capillary meanwhile the sample matrix remains in the pervaporator. By this approach volatile acidity and free sulfur dioxide have been simultaneously determined in wines. The detection limits (LODs) are 1.25 and 5.00 microg/mL, the quantification limits 4.12 and 16.50 microg/mL, and the linear dynamic ranges between LOD and 50 microg/mL and between 0.1 and 0.9 g/L for free sulfur dioxide and volatile acidity, respectively. The repeatability and within laboratory reproducibility, expressed as relative standard deviation (RSD), are 1.61% and 3.00% for free sulfur, and 3.35% and 4.58% for volatile acidity, respectively. The optimal pervaporation time and the time necessary for the individual separation-detection of the target analytes are 6 and 5 min, respectively. The analysis frequency is 7 h(-1) and the sample amount necessary is less than 7 mL. The proposed method and official methods for the analytes were applied to 32 wine samples. A two-tailed t-test was used to compare the methods, which yielded similar results. The errors, expressed as RSD for the two parameters, ranged between 1.3 and 4.1%.     

Characterization of NIST food-matrix standard reference materials for their vitamin C content

The vitamin C concentrations in three food-matrix Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST) have been determined by liquid chromatography (LC) with absorbance detection. These materials (SRM 1549a Whole Milk Powder, SRM 1849a Infant/Adult Nutritional Formula, and SRM 3233 Fortified Breakfast Cereal) have been characterized to support analytical measurements made by food processors that are required to provide information about their products’ vitamin C content on the labels of products distributed in the United States. The SRMs are primarily intended for use in validating analytical methods for the determination of selected vitamins, elements, fatty acids, and other nutrients in these materials and in similar matrixes. They can also be used for quality assurance in the characterization of test samples or in-house control materials, and for establishing measurement traceability. Within-day precision of the LC method used to measure vitamin C in the food-matrix SRMs characterized in this study ranged from 2.7 % to 6.5 %.     

Conformational study of glycyl-L-proline technetium complex

Quantitative measurement of fission and activation products resulting from neutron irradiation of fissile materials is of interest for applications in environmental monitoring, nuclear waste management, and national security. Based on established separation processes involving co-precipitation, solvent extraction, and ion-exchange and extraction chromatography, we have optimized a proposed sequence of separation steps to allow for the timely quantification of analytes of interest. We have recently evaluated this scheme using an irradiated sample to examine the adequacy of separations for measurement of desired analytes by gamma spectrometry. Here we present the radiochemical separations utilized and the yields and purity obtained.     

QuEChERS-超高效液相色谱-串联质谱法快速测定凉茶中非法添加的12种化学药物

建立了凉茶中马来酸氯苯那敏、吡罗昔康、α-细辛脑等12种非法添加的化学药物的QuEChERS结合超高效液相色谱-串联质谱(UPLC-MS/MS)的检测方法。样品经乙腈提取,应用QuEChERS技术净化,经0.22 μm微孔滤膜过滤后进行UPLC-MS/MS测定,以乙腈-0.1%(v/v)乙酸水溶液为流动相梯度洗脱,在XBridge BEH C₁₈柱(100 mm×2.1 mm, 3.5 μm)上实现12种化学药物的基线分离。该方法采用多反应监测(MRM)正离子模式扫描,标准曲线外标法定量。12种待测物在各自的质量浓度范围内线性关系良好,相关系数均大于0.997,检出限为0.1~2.1 μg/L,定量限为0.4~8.0 μg/L。在3个不同添加水平下的平均回收率为62.7%~95.2%, RSD为1.3%~10.8%。应用该方法对市面上销售的74批次凉茶进行了筛查测定,部分样品的测定结果为阳性。该方法操作简单,净化效果好,灵敏度高,适用于凉茶中12种非法添加的化学药物的快速分析。     

Determination of the active and inactive metabolites of prasugrel in human plasma by liquid chromatography/tandem mass spectrometry

Two fast and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bioanalytical assays were developed and validated to quantify the active and three inactive metabolites of prasugrel. Prasugrel is a novel thienopyridine prodrug that is metabolized to the pharmacologically active metabolite in addition to three inactive metabolites, which directly relate to the formation and elimination of the active metabolite. After extraction and separation, the analytes were detected and quantified using a triple quadrupole mass spectrometer using positive electrospray ionization. The validated concentration range for the inactive metabolites assay was from 1 to 500 ng/mL for each of the three analytes. Additionally, a 5x dilution factor was validated. The interday accuracy ranged from -10.5% to 12.5% and the precision ranged from 2.4% to 6.6% for all three analytes. All results showed accuracy and precision within +/-20% at the lower limit of quantification and +/-15% at other levels. The validated concentration range for the active metabolite assay was from 0.5 to 250 ng/mL. Additionally, a 10x dilution factor was validated. The interbatch accuracy ranged from -7.00% to 5.98%, while the precision ranged from 0.98% to 3.39%. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxyacetophenone immediately after collection was essential to ensure the stability of the metabolite during sample processing and storage. These methods have been applied to determine the concentrations of the active and inactive metabolites of prasugrel in human plasma.