Journal of Chromatography B (v.879, #2)

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50–100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10–50 kDa tangential flow filter.
Keywords: Hemoglobin; Protein purification; Immobilized metal ion affinity chromatography; Zinc;

Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry by Sirimas Sudsakorn; Abhishek Phatarphekar; Thomas O'Shea; Hanlan Liu (139-145).
Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D2 (1α(OH)D2), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D2 (1,25(OH)2D2). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC–MS/MS bioanalytical assays for 1,25(OH)2D2 suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC–MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0 pg/mL using 300 μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0–1000 pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100 ± 7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)2D2 rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D2.
Keywords: Vitamin D2; Bioanalysis; LC–MS/MS; Serum;

Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm × 4.6 mm i.d., 5 μm particle size) maintained at 40 °C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02–4 μg/mL, with correlation coefficients (r 2) ≥ 0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8 ± 12.5% (n  = 5), 83.5 ± 5.2% and 77.7 ± 2.0%, respectively (n  = 8 in both cases). The recovery for IS was 94.5 ± 7.9% (n  = 15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at −15 °C to −25 °C and −70 °C to −90 °C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.
Keywords: Ciprofloxacin; HPLC fluorescence detection; Plasma; Protein precipitation; Validation;

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA–DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C18 Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC–MS/MS with electrospray ionization in positive ion mode. The quantification of the AA–DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1 ± 0.3 dA-AAI per 109 normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as 32P-postlabelling and HPLC with fluorescence detection, the developed UPLC–MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.
Keywords: Aristolochic acid; DNA adduct; Ultra performance liquid chromatography–tandem mass spectrometry; Multiple reaction monitoring;

Detection of banned nitrofuran metabolites in animal plasma samples using UHPLC–MS/MS by Anita Radovnikovic; Mary Moloney; Paddy Byrne; Martin Danaher (159-166).
The use of nitrofurans as veterinary drugs in food-producing animals has been banned in the EU since the 1990s. Monitoring programs in the EU are based on the detection of protein-bound metabolites after slaughter. An UHPLC–MS/MS method was developed and validated for pre slaughter determination of four nitrofuran metabolites (AHD, AOZ, SEM, AMOZ) in animal plasma (bovine, ovine, equine and porcine). This method is proposed as an alternative method for on-farm surveillance. Plasma samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with organic solvent. Extracts were concentrated and then analysed by UHPLC–MS/MS. The method was validated according to Commission Decision 2002/657/EC. Inter-species recovery for AHD, AOZ, SEM and AMOZ was 72, 74, 57 and 71%, respectively. Decision limits (CCα) were calculated from within laboratory reproducibility experiments to be 0.070, 0.059, 0.071 and 0.054 μg kg−1, respectively. In addition, the assay was applied to incurred plasma samples taken from pigs treated with furazolidone.
Keywords: Nitrofuran metabolites; Plasma; Residue determination; UHPLC–MS/MS; Stability study;

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV by Roberto Mandrioli; Laura Mercolini; Domenico Lateana; Giancarlo Boncompagni; Maria Augusta Raggi (167-173).
Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery > 89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.
Keywords: Risperidone; 9-Hydroxyrisperidone; Plasma; Urine; Saliva; Micro-extraction by packed sorbent (MEPS);

Development of a liquid chromatography-tandem mass spectrometry with pressurized liquid extraction for determination of glucocorticoid residues in edible tissues by Dongmei Chen; Yanfei Tao; Zhaoying Liu; Huahai Zhang; Zhenli Liu; Yulian Wang; Lingli Huang; Yuanhu Pan; Dapeng Peng; Menghong Dai; Xu Wang; Zonghui Yuan (174-180).
A multi-residues method using pressurized liquid extraction (PLE) and liquid chromatography combined with mass spectrometry (LC–MS/MS) has been developed for determination of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, fludrocortisone) in muscle of swine, cattle, and sheep. Parameters affecting PLE extraction including extraction solvent, extraction temperature, extraction pressure and extraction cycles were optimized. The optimized method employed 11 ml extraction cells, hexane–ethyl acetate (50:50, v/v) as extraction solvent, 1500 psi of extraction pressure and 50 °C of extraction temperature. The samples were detected by LC-ESI-MS/MS in negative mode with selected reaction monitoring (SRM) mode. The recovery of glucocorticoids spiked at levels of 0.5–6 μg kg−1 ranged from 70.1% to 103.1%; the between-day relative standard deviations were no more than 9.6%. The limits of quantification were 0.5–2 μg kg−1 in muscle. The results demonstrated that the method is simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.
Keywords: Glucocorticoids; Liquid chromatography; Mass spectrometry; Pressurized liquid extraction; Residues; Edible tissues;

Molecularly imprinted solid-phase extraction for the selective determination of valnemulin in feeds with high performance liquid chromatography by Hongbin Guo; Kaiyong Liu; Yahong Liu; Binghu Fang; Min Liu; Limin He; Zhenling Zeng (181-185).
A simple, sensitive and reproducible high performance liquid chromatographic method was developed for determining valnemulin in feeds. Feed samples were extracted with ethyl acetate under alkaline condition, cleaned up by molecularly imprinted solid-phase extraction, and analyzed by high performance liquid chromatography with ultraviolet detection. The characteristics of the synthesized polymer were evaluated and the loading capacity of the polymer was about 1000 μg analyte/g imprinted polymer. The new procedure for the feed sample cleanup using the prepared polymer cartridge gave higher recoveries and fewer matrix interferences. The assay exhibited a linear dynamic range of 5.0–200 mg kg−1 with the correlation coefficient above 0.9993. Recoveries of valnemulin from feed samples spiked at 5.0, 20 and 50 mg kg−1 ranged between 76.0% and 94.4% with relative standard deviations of less than 9%. The limit of detection for valnemulin in feeds was 1 mg kg−1.
Keywords: Valnemulin; Feeds; High performance liquid chromatography; Molecularly imprinted polymer; Solid-phase extraction;

Human plasma quantification of droperidol and ondansetron used in preventing postoperative nausea and vomiting with a LC/ESI/MS/MS method by Jean-Claude Alvarez; Beny Charbit; Stanislas Grassin-Delyle; Jean-Louis Demolis; Christian Funck-Brentano; Emuri Abe (186-190).
An analytical method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the simultaneous identification and quantification of droperidol and ondansetron in human plasma. The two drugs were isolated from 0.5 mL of plasma using a basic liquid–liquid extraction with diethyl ether/heptane (90/10, v/v) and tropisetron and haloperidol as internal standards, with satisfactory extraction recoveries. They were separated on a 5-μm C18 Highpurity column (150 mm × 2.1 mm I.D.) maintained at 30 °C. The elution was achieved isocratically with a mobile phase of 2 mM HCOONH4 pH 3.8 buffer/acetonitrile (60/40, v/v) at a flow rate of 200 μL/min. Data were collected either in full-scan MS mode at m/z 100–450 or in full-scan MS–MS mode, selecting the [M+H] + ion at m/z  = 294.0 for ondansetron, m/z  = 285.2 for tropisetron, m/z  = 380.0 for droperidol and m/z  = 376.0 for haloperidol. The most intense daughter ion of ondansetron (m/z  = 212.0) and droperidol (m/z  = 194.0) were used for quantification. Retention times for tropisetron, ondansetron, droperidol and haloperidol were 2.50, 2.61, 3.10 and 4.68 min, respectively. Calibration curves were linear for both compounds in the 0.50–500 ng/mL range. The limits of detection and quantification were 0.10 ng/mL and 0.50 ng/mL, respectively. The intra- and inter-assay precisions were lower than 6.4% and intra- and inter-assay recoveries were in the 97.6–101.9% range for the three 3, 30 and 300 ng/mL concentrations. This method allows simultaneous and rapid measurement of droperidol and ondansetron, which are frequently co-administrated for the prevention of postoperative nausea and vomiting.
Keywords: Droperidol; Ondansetron; Postoperative nausea and vomiting; LC/ESI/MS/MS; Human plasma;

Selaginella sinensis (Selaginellaceae) is used extensively in traditional Chinese medicine (TCM) for the treatment of many kinds of chronic diseases. In this study, fractionation of the methanol extract of S. sinensis by different polarity solvents indicated the ethyl acetate fraction exhibited an potent 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity with the IC50 value of 44.9 μM. In order to evaluate the scientific basis, antioxidant peaks were firstly screened using DPPH spiking test through high performance liquid chromatography (DPPH-HPLC). Under the target-guidance of DPPH-HPLC experiment, two flavonoids and six biflavonoids, quercetin (1), apigenin (2), amentoflavone (3), robustaflavone (4), 2,3-dihydroamentaflavone (5), hinokiflavone (6), 4′-O-methyl-robustaflavone (7) and ginkgetin (8) were separated by high-speed counter-current chromatography (HSCCC) method using n-hexane–ethyl acetate–methanol–water (8:8:9:7) as the solvent system with purities 98.2%, 97.6%, 99.4%, 92.3%, 98.5%, 98.9% and 99.6%, respectively. The structures were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Antioxidant activity of eight isolated compounds was assessed by the radical scavenging effect on DPPH radical, compound 1 showed strongest antioxidant activities with IC50 values of 3.2 μM, while compounds 28 showed weak antioxidant activities. This is the first report on simultaneous separation of eight antioxidant compounds from S. sinensis by HSCCC, moreover, apigenin and 4′-O-methyl-robustaflavone were first identified from this plant. Results of the present study indicated that the combinative method using DPPH-HPLC and HSCCC could be widely applied for rapid screening and isolating of antioxidants from complex TCM extract.
Keywords: Selaginella sinensis; Antioxidant; DPPH-HPLC; HSCCC; Flavonoid; Biflavonoid;

Hollow fiber-based liquid phase microextraction (HF-LPME) for a highly sensitive HPLC determination of sulfonamides and their main metabolites by María Ramos Payán; Miguel Ángel Bello López; Rut Fernández-Torres; Mercedes Villar Navarro; Manuel Callejón Mochón (197-204).
In this paper, three phase-hollow fiber-based liquid phase microextraction (HF-LPME) combined with a HPLC procedure using diode array (DAD) and fluorescence detection (FLD) has been developed for the determination of four widely used sulfonamides: sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole and their main metabolites, the corresponding N4-acetyl derivatives: N4-acetyl-sulfadiazine, N4-acetyl-sulfamerazine, N4-acetyl-sulfamethazine, N4-acetyl-sulfamethoxazole. A Q3/2 Accurel KM polypropylene hollow fiber supporting 1-octanol was used between a 2 M Na2SO4 aqueous solution (pH 4) as a donor phase and aqueous solution (pH 12) as an acceptor phase. The procedure allows very low detection and quantitation limits of 0.3–33 ng L−1 and 0.9–100 ng L−1, respectively. The proposed method was applied to the determination of the analytes in environmental water samples (surface, tap and wastewater).
Keywords: Hollow fiber liquid phase microextraction; HF-LPME; Sulfonamides; Metabolites; Environmental water; HPLC;

A new method of analysis has been developed and validated for the determination of firocoxib, a new nonsteroidal anti-inflammatory drug (NSAID) approved for use in horses and dogs to control pain and inflammation associated with osteoarthritis. Following a liquid extraction using ethyl acetate:hexane (40:60), samples were separated by isocratic reversed-phase HPLC on a Sunfire C18 column and quantified using UV detection at 290 nm. The mobile phase was a mixture of water with 0.025% trifluoroacetic acid and acetonitrile, with a flow-rate of 1.1 ml/min. The procedure produced a linear curve over the concentration range 5–1500 ng/ml with a lower limit of quantification of 5 ng/ml. Intra- and inter-assay variability was less than 7%. The average recovery was 98%. The method is suitable for the analysis of clinical samples from pharmacokinetic studies and can also be used for small volume sample sizes.
Keywords: Analgesic; Firocoxib; HPLC; NSAID; Pharmacokinetics;

GC–MS analysis of bisphenol A in human placental and fetal liver samples by Jie Zhang; Gerard M. Cooke; Ivan H.A. Curran; Cynthia G. Goodyer; Xu-Liang Cao (209-214).
A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography–mass spectrometry (GC–MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. β-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings.
Keywords: Bisphenol A; Human fetal liver; Human placenta; Gas chromatography; Mass spectrometry;

HPLC MS/MS method for quantification of meprobamate in human plasma: Application to 24/7 clinical toxicology by Xavier Delavenne; Jean Pierre Gay-Montchamp; Thierry Basset (215-218).
We described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of meprobamate in human plasma with [13C-2H3]-meprobamate as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. Separation was performed by reversed-phase chromatography on a Luna MercuryMS C18 (20 mm × 4 mm × 3 μm) column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The selected reaction monitoring transitions, in electrospray positive ionization, used for quantification were 219.2 → 158.2 m/z and 223.1 → 161.1  m/z for meprobamate and internal standard, respectively. Qualification transitions were 219.2 → 97.0 and 223.1 → 101.1 m/z for meprobamate and internal standard, respectively. The method was linear over the concentration range of 1–300 mg/L. The intra- and inter-day precision values were below 6.4% and accuracy was within 95.3% and 103.6% for all QC levels (5, 75 and 200 mg/L). The lower limit of quantification was 1 mg/L. Total analysis time was reduced to 6 min including sample preparation. The present method is successfully applied to 24/7 clinical toxicology and demonstrated its usefulness to detect meprobamate poisoning.
Keywords: Meprobamate; LC–MS/MS; Poisoning;

Ursolic acid is a hydroxy pentacyclic triterpene, which proved to have sedation, anti-inflammatory, antibacterial, antiulcer and anti-cancer activities. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method with high selectivity, sensitivity and throughput has been established and validated for quantitation of total ursolic acid in human plasma. Plasma samples were pretreated by liquid–liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C8 column (100 mm × 2.1 mm, I.D., 1.7 μm) using mobile phase consisting of acetonitrile and 10 mM ammonium formate (90:10, v/v) at 0.2 mL/min. The duration of chromatography analysis was 3 min. The multiple reaction monitoring (MRM) was performed at m/z 455.1 → 455.0 for ursolic acid and m/z 469.3 → 425.2 for glycyrrhetinic acid (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The assay showed good linearity over the range of 10–5000 ng/mL for ursolic acid in human plasma with a lower limit of quantitation of 10 ng/mL. The mean extraction recovery was 73.2 ± 4.5% and the matrix ion suppression ranged from −11.4% to −5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study after intravenous infusion of Ursolic Acid Nano-liposomes to healthy volunteers.
Keywords: Ursolic acid; UPLC/MS/MS; Clinical pharmacokinetics;

Determination of anabolic steroids in bovine serum by liquid chromatography–tandem mass spectrometry by George Kaklamanos; Georgios A. Theodoridis; Themistoklis Dabalis; Ioannis Papadoyannis (225-229).
In the present paper we report the LC–MS/MS determination of residues of 12 anabolic steroids in bovine serum, as an expansion of our work protocols for steroids determination in biological matrices. Steroids analyzed included α-zearalanol, β-zearalanol, α-trenbolone, β-trenbolone, methyltestosterone, α-estradiol, β-estradiol, ethynylestradiol, α-boldenone, β-boldenone, α-nortestosterone and β-nortestosterone. Following protein precipitation, serum samples were cleaned up by solid-phase extraction using Oasis HLB and Amino cartridges. Atmospheric pressure chemical ionization (APCI) in both positive and negative ionization modes was used and mass spectrometry detection was carried out in multiple reaction monitoring mode following two or (in most cases) three product ions per precursor ion. The method was validated in accordance with the Commission Decision 2002/657/EC. The decision limit (CCα) values obtained, ranged from 0.01 to 0.07 ng/ml and the detection capability (CCβ) values obtained ranged from 0.02 to 0.12 ng/ml. The recoveries ranged from 70.2% to 118.2%. The developed method is suitable for routine and confirmatory purposes such as control of illegal use in livestock production.
Keywords: Anabolic steroids; Serum; Validation; Liquid chromatography–tandem mass spectrometry (LC–MS/MS);