Direct and rapid characterization of illicit drugs in adulterated samples using thermal desorption electrospray ionization mass spectrometry

Foods and drinks have been adulterated with illicit drugs to facilitate criminal activities. Unfortunately, conventional analytical methods are incapable of rapidly characterizing these drugs in samples, as serious interferences from sample matrices must be removed through tedious and time-consuming pretreatment. Ambient ionization mass spectrometry (AMS) generally does not require sample pretreatment and is thus a suitable tool for directly and rapidly detecting illicit drugs in samples in different physical states. In this study, thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS), an AMS technique, was utilized to efficiently characterize illicit drugs spiked in samples including drinks, powders, and jelly candies. To perform sensitive analysis, the mass analyzer was operated in multiple reaction monitoring mode to monitor the molecular and fragment ions of the target analytes. The time required to complete a typical TD-ESI/MS analysis was less than 30 s. The limits of detection (LODs) for illicit drugs were found to be 100 ppb in drinks, 100–1000 ppb in instant powders, and 1.3–6.5 ng/mm² on stamp surfaces. FM2 and nitrazepam laced in the inner layer of a jelly candy were detected by TD-ESI/MS, showcasing the advantage of the technique for direct and rapid analysis as opposed to conventional methods.     

An ultra-rapid drug screening method for acetaminophen in blood serum based on probe electrospray ionization-tandem mass spectrometry

Poisoning incidents caused by drugs, accidental ingestion of poisons, attempted suicide, homicide, and exposure to toxic compounds occur frequently every year across the globe. This raises the need to rapidly identify toxic agents in poisoned patients in a clinical emergency setting. In addition, determining drug/poison concentration is undoubtedly necessary to arrive at a toxicological treatment plan. The purpose of this study was to develop an ultra-rapid drug screening method for the clinical treatment of poisoning. Probe electrospray ionization (PESI), one of the ambient ionization techniques, is able to detect compounds from various biological materials almost directly. We applied the PESI technique to the rapid detection of acetaminophen (APAP). Blood serum samples were diluted 100-fold with 10 mM ammonium formate/ethanol (1:1 v/v) solution including deuterium-labeled internal standards (IS; APAP-d4). Only 10 μL of the diluted sample was used for measurement. The tandem mass spectrometer (MS/MS) equipped with a PESI was used in selected reaction monitoring mode for the quantitation of APAP; the measurement time was only 18 s. Transitions were set at 152 > 110 for quantitation, 152 > 65 for qualifier, and 156 > 114 for IS (APAP-d4). All measurements were conducted in positive mode. The calibration curve (1/x²) was linear over the range of 1.56–200 μg/mL (r² = 0.998), and the limit of detection and quantitation were 0.37 μg/mL and 1.56 μg/mL, respectively. The accuracy (bias) and precision (RSD%) of the method were within an acceptable range (−0.15–2.8% and 2.3–6.1%, respectively) and matrix effect at 3 concentrations (95.1–104%) indicated that PESI-MS/MS is only slightly affected by matrices. In real forensic cases, quantitative values of APAP determined by the PESI-MS/MS were almost identical to those determined by the liquid chromatography-MS/MS method. Since PESI-MS/MS is a simple, reliable, and rapid determination method for toxic agents with virtually no need for blood serum pretreatment, it would be highly suitable for poisoning cases in clinical emergency settings. In the future, a method for simultaneous rapid determination of multiple toxic agents will be developed.     

D-赖氨酸、L-谷氨酰胺和L-酪氨酸对格列美脲及其顺式异构体识别的电喷雾质谱法研究

目的 使用电喷雾质谱技术,研究3种氨基酸（D-赖氨酸、L-谷氨酰胺和L-酪氨酸）作为手性选择探针对格列美脲和格列美脲异构体的识别效应。方法 分别将3种氨基酸与格列美脲及其顺式异构体溶液混合,用电喷雾/离子阱质谱提取非共价结合的复合物,在二级质谱中通过碰撞诱导解离（CID）方法给予不同的碎裂能量,考察碎片离子的丰度从而进行格列美脲及其顺式异构体的识别。结果 L-酪氨酸、D-赖氨酸和L-谷氨酰胺对格列美脲及其顺式异构体均有识别效应,识别率分别为1.61、2.92和2.17。结论 以3种手性氨基酸作为立体异构区分探针,首次实现了格列美脲和格列美脲顺式异构体的质谱法快速识别。     

Styrene oxide DNA adducts: in vitro reaction and sensitive detection of modified oligonucleotides using capillary zone electrophoresis interfaced to electrospray mass spectrometry

Styrene is one of the most important synthetic chemicals in the world and is subject to investigations concerning carcinogenicity and mutagenicity due to the active metabolite, styrene-7,8-oxide. This epoxide shows a tendency to react, among others, with DNA and DNA constituents. The in vitro reaction of styrene oxide with DNA was investigated by cleaving incubated calf thymus DNA with two different enzymes, namely Benzonase and alkaline phosphatase, to obtain oligonucleotides of the type n-nucleotide-(n− 1)-phosphate with chain length from 2 to 8 bases. Alkylated and non-alkylated nucleotides were separated in groups according to their chain length using capillary zone electrophoresis and were detected with electrospray mass spectrometry. This improvement in sensitivity made it possible to obtain new information about the reaction of styrene oxide with DNA, especially to detect unknown reaction products. The results indicate that primarily purine bases were alkylated by styrene oxide before pyrimidine bases, which react with higher concentrations of styrene oxide. This means that in addition to the already reported adducts in DNA at the N-7-, O⁶- and N²-position of guanine also adducts at the nucleophilic sites of adenine can be found using mass spectrometry. We anticipate for the future this procedure will allow us to investigate base sequence specific reactions as well as interactions from xenobiotics and cytostatic drugs, since reaction products would directly be detectable. Received: 16 January 1997 / Accepted: 17 March 1997     

A rapid, simple, specific liquid chromatographic-electrospray mass spectrometry method for the determination of finasteride in human plasma and its application to pharmacokinetic study

A fast, accurate, sensitive, selective and reliable method using reversed-phase high-performance liquid chromatography-mass spectrometry coupling with an electrospray ionization interface was developed and validated for the determination of finasteride in human plasma. After deprotienation with acetonitrile, centrifugation, evaporation to dryness and dissolving in mobile phase, satisfactory separation was achieved on a Hypersil-Keystone C(18) reversed-phase column using a mobile phase consisting of acetonitrile-water (46:54, v/v), 0.1% acetic acid and 0.1% trifluoracetic acid. Carbamazepine (IS) was used as internal standard. This method involved the use of the [M+H](+) ions of finasteride and IS at m/z 373 and 237 with the selective ion monitoring (SIM) mode. The calibration curve was linear in the range of 0.2-120 ng ml(-1). The limit of quantification for finasteride in plasma was 0.2 ng ml(-1) with good accuracy and precision. The intra-assay precision and accuracy were in the range of 2.1-11.2% and -1.3% to 8.5%, respectively. The inter-assay precision and accuracy were in the order of 3.4-12.1% and -1.5% to 11.5%, respectively. The mean sample extract recoveries of the method were higher than 85% and 74% for finasteride and internal standard (IS), respectively. The assay has been successfully used to estimate the pharmacokinetics of finasteride after oral administration of a 5mg tablet of finasteride to 24 healthy volunteers.     

应用H/D交换质谱法对庆大霉素C2/C2a及小诺霉素进行快速鉴别

目的快速鉴别庆大霉素C2/C2a和小诺霉素。方法采用电喷雾质谱法结合氢-氘（H/D）交换技术对庆大霉素C2/C2a和小诺霉素进行研究。结果进行氘代反应后,庆大霉素C2/C2a和小诺霉素的m/z可分别得到＋13和＋12的增量。结论上述方法可以简便快速地区分出庆大霉素C2/C2a与小诺霉素这一对同分异构体,为鉴别分子内活泼氢数目不同的同分异构体提供了一种可行的方法。     

A rapid and simple ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method for the detection of glucuronic acid-conjugated steroid metabolites

In the present study, we effectively detected 10 steroids and glucuronic acid-conjugated steroid metabolites in 12 min by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Steroids testosterone (T), 5α-dihydrotestosterone (DHT), androsterone (ADT), etiocholanolone (ETIO), estradiol (E₂) and their glucuronide conjugates were well-separated on an Eclipse Plus C₁₈ column (2.1 mm×50 mm, RRHD 1.8 µm). The mobile phase consisted of a mixture of methanol and ultrapure water (containing 1 mM ammonium formate) at a ratio of 60:40 (v/v), and the flow rate was set at 0.25 mL/min. The LC eluate was detected by electrospray ionization (ESI) source in both positive and negative ion modes. Neutral loss (NL of 176, 194, 211 and 229 Da in positive mode) and precursor ion (PI of m/z 141, 159 and 177 in positive mode and 75, 85 and 133 in negative mode) methods were applied for the detection of steroid glucuronides. The multiple reaction monitoring (MRM) transitions were m/z 289.3→97.1, 291.3→105, 291.3→199.2, 273.2→145.4 and 255.2→159.1 for T, DHT, ADT, ETIO and E₂ in positive mode, respectively; as well as m/z 463.3→85 for T glucuronide (T-G), m/z 465.3→75 for DHT glucuronide (DHT-G), ADT glucuronide (ADT-G), ETIO glucuronide (ETIO-G) and m/z 447.3→271 for E₂ glucuronide (E₂-G) in negative mode. In addition, the analytical method was also applied for the detection of steroid glucuronides in pooled human liver microsomes (HLM), which might serve as a basis for further investigation of steroid metabolism in vivo and in vitro.     

Development and validation of a simple and sensitive liquid chromatography–tandem mass spectrometry method for quantifying trimetazidine in human plasma

1. A simple and sensitive liquid chromatography–tandem mass spectrometry (LC‐MS‐MS) method for quantifying trimetazidine in human plasma was developed and validated. Sample preparation was based on deproteinating with acetonitrile. 2. Chromatography was performed on a C18 analytical column (5 μm; 150 × 2.1 mm i.d.) and the retention times for trimetazidine and cetirizine (used as the internal standard) were 1.8 and 3.0 min, respectively. The ionization was optimized using an electrospray ionization source and enhanced selectivity was achieved using tandem mass spectrometry. The calibration curve ranged from 0.1 to 200 ng/mL. The inter‐day precision, accuracy and the relative standard deviation (RSD) were all < 15%. The analyte was shown to be stable over the time‐scale of the entire procedure. 3. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.     

LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

高分离度快速液相色谱-三重串联四极杆质谱快速检查半夏和虎掌南星中水麦冬酸

目的 建立半夏及其伪品虎掌南星中水麦冬酸的专属性检测方法。方法 样品经水超声提取,利用高分离度快速液相色谱-三重四极杆质谱（rapid resolution liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry,RRLC-QQQ/MS）检测水麦冬酸。采用Agilent SB-C₁₈（2.1 mm×50 mm,1.8 μm）色谱柱,以乙腈-0.1%甲酸（5∶95）为流动相。柱温30℃,体积流量0.2 mL/min,进样量1 μL。离子化模式为ESI^-,选择m/z187→143和m/z187→99作为检测离子对,进行多反应监测。结果 检出限为4.6 μg/L。11批半夏中有3批检出水麦冬酸,7批虎掌南星均检出水麦冬酸,半夏中水麦冬酸含量低于虎掌南星。结论 所建方法专属、准确、快速、简便,可为半夏的质量控制提供参考。     

Development and validation of a method for quantitative determination of valsartan in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of valsartan in human plasma was developed and validated. A 0.5 ml aliquot was extracted using solid-phase extraction in an Empore^® high performance extraction disk plate, universal resin 96-well format. The estimated calibration range of the method was 2–2000 ng/ml.

The method was fully validated with intra-day mean accuracy and precision of 94.8–107% and 2.19–5.40% and inter-day mean accuracy and precision of 93.5–105% and 1.87–5.67%, respectively. No significant loss of valsartan in processed samples was confirmed in processed samples for up to 24 h at 10 °C. Sample dilution up to 50-fold with blank human plasma provided acceptable analyses. No interference peaks or matrix effects were observed. No effect of QC sample location results was observed in a 96-well plate. This LC-MS/MS technique was found to improve quantitative determination of valsartan allowing its pharmacokinetic evaluation with clinically relevant doses.     

Development and validation of a high-performance liquid chromatography-electrospray mass spectrometry method for the simultaneous determination of 23 eicosanoids

Inflammation is implicated in the pathogenesis of a number of diseases, including cardiovascular disease. Current research is focused on developing assays to search for biomarkers for inflammation. Eicosanoids are the oxidative metabolites of arachidonic acid (eicosatetraenoic acid, AA), a long chain polyunsaturated fatty acid common in Western diets. AA can be oxidized by one of three pathways to form prostaglandins (PGs), leukotrienes (LTs), or a number of hydroxyl and epoxy compounds. These eicosanoids have a variety of physiological functions, including regulating inflammation. We have developed a method utilizing LC-MS to separate and quantitate 23 different eicosanoids from all the three oxidative pathways. The eicosanoids were separated using a gradient elution of acetonitrile with 0.1% formic acid (v/v) and water with 0.1% formic acid (v/v) at a flow rate of 1 mL/min with a Symmetry C18 column (250 mm x 4.6 mm). Deuterated eicosanoids were used as internal standards for quantitation. Mass spectrometric detection was carried out using an Agilent 1100-series LC-MSD with an electrospray ionization interface. Electrospray ionisation (ESI) mass spectra were acquired using negative ionization and selective ion monitoring. The method was validated and shown to be sensitive (LOQ at pg levels for most compounds), accurate (recovery values 75-120%) and precise (R.S.D.<20 for all compounds) with a linear range over several orders of magnitude. The method was applied to rat kidney tissue and shown to be indicative of the eicosanoid levels within a specific organ. The analysis of eicosanoids can provide insight into the inflammatory mechanisms associated with cardiovascular disease.     

Insights into the neutral loss of 30 Da of Paeonia monoterpene glycosides in electrospray ionization tandem mass spectrometry and the rapid screening of monoterpene glycosides by LC/MS/MS

Monoterpene glycosides are the major bioactive compounds of Paeonia lactiflora Pall (P. lactiflora). Characteristic neutral loss of 30 Da has been extensively reported for monoterpene glycosides in tandem mass spectrometry. However, little is known about mechanism of this fragmentation. The neutral loss of 30 Da was studied for eleven monoterpene glycosides (1–11) from P. lactiflora by ion trap mass spectrometry in this report. Compounds 1–5 with a hemiacetal structure could readily lose 30 Da at low collision energy of 30% in MS/MS by ion trap mass spectrometry. For compounds 6–11, neutral loss of 30 Da could also be observed at low abundance, but the collision energy had to be increased to 60%. In both cases, high-accuracy mass spectrometry assigned the 30 Da as CH₂O. After careful analysis of the structures and mass spectra, we believe that the neutral loss of 30 Da in compounds 1–5 was due to cleavage of the hemiacetal structure, whereas it was ascribed to the cleavage of 5¢-hydroxymethyl group of the glucosyl residue in other monoterpene glycosides. Furthermore, the characteristic neutral loss of 30 Da at low collision energy was used to screen hemiacetals from crude extracts of P. lactiflora and related plant species. Significant differences among Paeonia species were observed by 30 Da neutral loss analysis.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5μm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.     

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine

In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC‐C‐IRMS) analysis is performed. Recently published work suggests that epiandrosterone sulfate (EpiAS) is a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form, while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean‐up. The current work presents a new, fast, and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non‐hydrolyzed EpiAS, making the solid phase extraction, hydrolysis, and acetylation step redundant. Sample preparation consisted of a simple liquid–liquid extraction, followed by LC fraction collection. A population study was performed to check compliance with the criteria drafted by the World Anti‐Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e. dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION), and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as the target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.     

Development and validation of a simple online‐SPE method coupled to high‐resolution mass spectrometry for the analysis of stanozolol‐N‐glucuronides in urine samples

Stanozolol is still the most commonly used illicit anabolic‐androgenic steroid (AAS) in professional sports. Therefore, accurate and fast analysis and long detection windows are of great interest in the field of antidoping analysis. In this work, a very simple, fast, and highly sensitive online solid‐phase extraction method coupled with liquid chromatography–high‐resolution tandem mass spectrometry (HPLC‐HRMSMS) for the analysis of stanozolol‐N‐glucuronides was developed. This fully validated procedure is characterized by only a few manual steps (dilution and addition of internal standard) in the sample preparation. A limit of identification (LOI) of 75 pg/mL, high accuracy (87.1%–102.1%), precision (3.1%–7.8%), and sensitivity was achieved. Furthermore, good linearity (> 0.99) and robustness, as well as no carry‐over effects, could be observed. In addition to excellent confirmation analysis performance, this method shows sufficient potential for the identification and characterization of unknown metabolites. Using this method, it was possible to unambiguously confirm the presence of 1′N‐ and 2′N‐stanozolol‐glucuronide in human urine for the first time due to the access to reference material.     

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N=3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N=10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05-10nM for ACh, 0.25-50 nM for iso-ACh and 15-3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.     

A sensitive and rapid liquid chromatography-tandem mass spectrometry method for the quantification of the novel neurokinin-1 receptor antagonist aprepitant in rhesus macaque plasma,and cerebral spinal fluid,and human plasma with application in translational NeuroAIDs research

A sensitive and rapid liquid chromatography-tandem mass spectrometry method has been developed for to assess therapeutic exposures of aprepitant in HIV-infected patients and rhesus macaques. The method utilized a simple sample-preparation procedure of protein precipitation with methanol. Chromatographic separation was performed on a reversed phase C₈ column (Hypersil Gold, 50 mm × 2.1 mm, 3 μm) using a mobile phase composed of acetonitrile and water in 0.5% formic acid through gradient elution. Electro-spray ionization in positive mode was incorporated in the tandem mass spectrometric detection. The lower limit of quantitation of aprepitant in plasma of rhesus macaques and human and cerebral spinal fluid of rhesus macaques were 1, 1, and 0.1 ng/mL, respectively. The method has been successfully employed to measure aprepitant in preclinical and clinical samples collected from three SIV-infected rhesus macaques and ten patients with HIV infection. In conclusion, this liquid chromatography-tandem mass spectrometry method is suitable for preclinical–clinical translational research exploring exposure–response relationships with aprepitant as well as therapeutic drug monitoring of aprepitant.     

Optimization,validation, and comparison of a rapid method for the quantification of insulin-like growth factor 1 in serum using liquid chromatography–high-resolution mass spectrometry

Human insulin-like growth factor 1 (IGF-I) is the primary mediator of the effects of the growth hormone (GH). Therefore, it has been used as a biomarker to detect the abuse of GH in sports. The measurement of IGF-I relies on mass-based and immunological approaches to analysis. Among the mass-based analysis methods, liquid chromatography–mass spectrometry (LC–MS) has a number of functional advantages. LC–MS measurements based on the quantification of IGF-I, according to trypsin digestion, are used in the most common method of analyzing doping. However, this method is time-consuming and subject to experimental variability. In this study, we optimized a rapid method for detecting IGF-I without the trypsin digestion step. This method of analysis uses an ultra-centrifugal filter and an LC-HRMS through narrow-range mass scan method. To verify the validity of this method, eight categories of validation testing were applied with the following results: linearity, R² > 0.99; limit of detection, 15 ng/ml; limit of quantification, 20 ng/ml; accuracy, >99%; recovery rate, >95%; carryover, <0.03; and inter- and intra-day precision values, %CV < 2% and %CV < 6%, respectively. Furthermore, we discussed the correlation of the quantified concentration from two other methods, immunoradiometric assay (IRMA) and parallel reaction monitoring method, using 209 serum samples. In conclusion, although both mass spectrometry-based methods worked equally well in terms of analytical performance and correlation with IRMA results, narrow-range mass scan method had several advantages, such as time and cost savings and reliable reproducibility, over the existing methods.     

Detecting the abuse of 19-norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19-norandrosterone and 19-noretiocholanolone

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography–tandem mass spectrometry (GC–MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with β-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ¹³C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of “unstable” urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.