Journal of Chromatography B (v.970, #C)

The aim of the study was to develop and validate a simple, sensitive and specific method for the detection and quantitative determination of 88 substances among psychoactive drugs and their metabolites in whole blood, and to apply the procedure to postmortem cases. Samples were consecutively diluted with methanol, acetonitrile and mobile phase. All the molecules were separated and then identified through a liquid chromatographic, tandem mass spectrometric system, and eventually fully validated according to the international guidelines. The method proved to be highly sensitive and specific and all the validation parameters fulfilled the acceptance criteria. In particular linearity was studied in the range LOQ-1000 ng/mL; matrix effects and carry over were negligible and the majority of the compounds assessed to be stable over several freeze and thaw processes. Olanzapine is the most unstable compound. Protryptiline and flupenthixol did not fulfilled acceptance criteria, and although their transitions were kept on the instrumental settings, they were not considered for the fully validation. The method was applied to several postmortem cases, and the results were compared to the GC–MS systematic toxicological analysis currently in use in our laboratory, assessing to be a good complementary procedure and providing a better sensitivity. The LC–MS/MS method could be easily applicable to routine analyses of postmortem samples, as well as to a screening procedure for clinical purposes; however it should be carried out in combination with a general unknown screening method.
Keywords: Blood; Psychoactive drugs; LC–MS/MS; Postmortem;

Two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to study the excretion and metabolic interaction of edaravone and taurine in rats by Dao-quan Tang; Xiao-xiao Zheng; Yin-jie Li; Ting-ting Bian; Yan-yan Yu; Qian Du; Dong-zhi Yang; Shui-shi Jiang (8-17).
In this study, two independent and complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were respectively developed and validated for the determination of edaravone or taurine in rat urine, feces and bile after intravenous administration, using 3-methyl-l-p-tolyl-5-pyrazolone and sulfanilic acid as the internal standards (IS). Edaravone was separated on an Agilent Eclipse Plus C18 column (100 × 2.1 mm, 3.5 μm) using methanol and water (containing 5 mM ammonium formate and 0.02% formic acid) as mobile phase, while taurine was performed on a Waters Atlantis HILIC Silica column (150 × 2.1 mm, 3 μm) using acetonitrile and water (containing 5 mM ammonium formate and 0.2% formic acid) as mobile phase. The mass analysis was performed in a Triple Quadrupole mass spectrometer via multiple reaction monitoring (MRM) with negative ionization mode. The optimized mass transition ion pairs (m/z) for quantification were 173.1→92.2 and 187.2→106.0 for edaravone and its IS, 124.1→80.0 and 172.0→80.0 for taurine and its IS, respectively. The validated methods have been successfully applied to the excretion and metabolism interaction study of edaravone and taurine in rats after independent intravenous administration and co-administration with a single dose. The results demonstrated that there were no significant alternations on the metabolism and cumulative excretion rate of edaravone and taurine, implying that the proposed combination therapy was pharmacologically viable.
Keywords: Edaravone; Taurine; Excretion; Metabolism; LC-MS/MS;

LC–MS metabolite fingerprinting and MtSK-based screening of an endophyte Bartalinia pondoensis Marinc of Citrus aurantum L by Ahmed M. Zaher; Makboul A. Makboul; Ahmad M. Moharram; Angela I. Calderón (18-23).
An endophyte Bartalinia pondoensis Marinc of Citrus aurantum L. var. dulcis was isolated and studied for its secondary metabolites and for their Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitory activities. Using LC–MS metabolite fingerprinting of the constituents of the methanol extract, 19 compounds pertaining to various classes were identified: amino acids, proto-alkaloids, fatty acid amides and oxazole, aniline derivatives and aromatic compounds. We report here for the first time the presence of the [N-(ethyloxy, hydroxymethyl)phenylethylamine] as a new proto-alkaloid and 18 other known compounds are reported for the first time in the genus of Bartalinia. MtSK inhibitory activities of methanol extract and fractions obtained by solid phase extraction (SPE) at a concentration of 50 μg/mL may be attributed to the presence of aniline and oxazole derivatives present in all fractions in varying concentrations.
Keywords: LC–MS; Bartalinia pondoensis; Metabolic fingerprinting; Endophyte;

An HPLC–MS/MS method for simultaneously determination of the active metabolites (67M-1, 67M-2 and 67M-4) in human plasma using clopidogrel as the internal standard was developed and validated. The compounds were extracted by protein precipitation using acetonitrile and separated using a C8 column by a gradient elution with the mobile phase consisting of acetonitrile (containing 0.1% formic acid) and 0.1% formic acid. Quantification was performed using multiple reaction monitoring in positive mode with m/z transitions of 333.1–261.0, 333.1–261.0, 347.0–261.0 and 322.2–184.1 for 67M-1, 67M-2, 67M-4 and clopidogrel (Internal Standard), respectively. This method was validated in terms of specificity, linearity, precision, accuracy, and stability. The lower limit of quantification of this method was 0.5 ng/mL and the calibration curve was linear over the concentration range of 0.5–150 ng/mL. The intra- and inter-run precision was less than 11.67% and 8.64%, respectively, with the accuracy between 98.33% and 108.38%. The samples were stable under all the tested conditions. This method has been successfully applied to the pharmacokinetic study of febuxostat in healthy Chinese volunteers following oral administration of 40 mg and 80 mg febuxostat.
Keywords: Febuxostat; Active metabolites; HPLC–MS/MS; Human plasma; Pharmacokinetics;

A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5 μL injected) were analyzed with selected reaction monitoring liquid chromatography–tandem mass spectrometry (LC–MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100 mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50–5000 pg/mL with and LOD 24 pg/mL. Calibration response showed good linearity (correlation coefficients < 0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000 pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n  = 95).
Keywords: Budesonide; Human plasma; LC–MS/MS; Phospholipid removal; Solid phase extraction; Automation;

Improved stability of TMS derivatives for the robust quantification of plant polar metabolites by gas chromatography–mass spectrometry by Anthony Quéro; Cyril Jousse; Michelle Lequart-Pillon; Eric Gontier; Xavier Guillot; Bernard Courtois; Josiane Courtois; Corinne Pau-Roblot (36-43).
Plant metabolite profiling is commonly carried out by GC–MS of methoximated trimethylsilyl (TMS) derivatives. This technique is robust and enables a library search for spectra produced by electron ionization. However, recent articles have described problems associated with the low stability of some TMS derivatives. This limits the use of GC–MS for metabolomic studies that need large sets of qualitative and quantitative analyses. The aim of this work is to determine the experimental conditions in which the stability of TMS derivatives could be improved. This would facilitate the analysis of the large-scale experimental designs needed in the metabolomics approach. For good repeatability, the sampling conditions and the storage temperature of samples during analysis were investigated. Multiple injections of one sample from one vial led to high variations while injection of one sample from different vials improved the analysis. However, before injection, some amino acid TMS derivatives were degraded during the storage of vials in the autosampler. Only 10% of the initial quantity of glutamine 3 TMS and glutamate 3 TMS and 66% of α-alanine 2 TMS was detected 48 h after derivatization. When stored at 4 °C until injection, all TMS derivatives remained stable for 12 h; at −20 °C, they remained stable for 72 h. From the integration of all these results, a detailed analytical procedure is thus proposed. It enables a robust quantification of polar metabolites, useful for further plant metabolomics studies using GC–MS.
Keywords: GC–MS; Metabolite profiling; Amino acids; TMS derivatives;

A specific, sensitive and fast LC–MS/MS method with positive electrospray ionization for the quantitative determination of nitrite in human plasma is reported. Added [15N]nitrite served as the internal standard (IS). Endogenous nitrite and IS were converted to their S-nitrosoglutathione (GSNO) derivatives, i.e., GS14NO and GS15NO, respectively, by using excess glutathione (GSH) and HCl. For plasmatic nitrite, fresh plasma (0.5 mL) was spiked with the IS (1000 nM) and ultrafiltered (cut-off 10 kDa). Ultrafiltrate aliquots (100 μL) were treated with aqueous GSH at a final concentration of 1 mM and 1 μL of 5 M HCl for 5 min. After final sample dilution (1:1, v/v) with acetonitrile–water (70:30, v/v), 2 μL aliquots were injected via a thermostated (4 °C) autosampler. The mobile phase was acetonitrile–water (70:30, v/v), contained 20 mM ammonium formate, had a pH value of 7, and was pumped isocratically at 0.5 mL/min. A Nucleoshell column was used for LC separation. The retention time of GSNO was about 0.8 min and the total analysis time 5 min. Quantification was performed by selected-reaction monitoring the specific mass transition m/z  337 ([M+H]+) →  m/z 307 ([M+H−14NO]+ •) for GS14NO (i.e., for endogenous nitrite) and m/z  338([M+H]+) →  m/z  307 ([M+H−15NO]+ •) for GS15NO (i.e., for the IS). The method was thoroughly validated in human plasma (range, 0–2000 nM). The LOD and LOQ values of the LC–MS/MS method were determined to be 1 fmol and 5 nM [15N]nitrite, respectively. The relative matrix-effect of about 21% was outweighed entirely by the IS. In freshly prepared plasma samples from heparinized blood donated by three healthy subjects, nitrite concentration was determined by LC–MS/MS to be 516, 199 and 369 nM. These concentrations were confirmed by using a previously reported GC–MS method and agree with those measured previously by HPLC–UV (334 nm) after nitrite conversion to S-nitroso-N-acetylcysteine (SNAC) by N-acetylcysteine (NAC). Measurement of nitrite by LC–MS/MS as GSNO is about 1000 times more sensitive than by HPLC–UV as SNAC. The applicability of the method to microdialysate, urine, and saliva samples from humans was demonstrated. The agreement of two orthogonal MS-based methods indicates that the concentration of nitrite in freshly prepared, non-frozen plasma from heparinized blood of fasted healthy humans is of the order of 400 nM.
Keywords: Derivatization; GC–MS; Glutathione; Matrix-effects; Nitric oxide; S-Nitrosylation;

The present study describes a novel liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the simultaneous estimation of ramipril (RAM) and hydrochlorothiazide (HCTZ) in human plasma using liquid–liquid extraction technique. This method made use of electrospray ionization in positive mode for RAM and in negative mode for HCTZ using triple quadrupole mass spectrometry where carbamazepine was used as an internal standard (IS). Analytes were recovered by methyl tertiary butyl ether:dichloromethane (85:15) subsequently separated on an Enable C18 G column (150 mm × 4.6 mm, 5 μm) using methanol:0.1% formic acid in water (85:15) as a mobile phase, at a flow rate of 0.5 mL/min. Quantification of RAM, HCTZ and IS was performed using multi-reaction monitoring mode (MRM) where transition of m/z 417.2 → 234.1 (RAM) and 237.0 → 194.0 (IS) in positive mode and 296.1 → 205.0 for HCTZ in negative mode. The calibration curve was linear (r 2  > 0.99) over the concentration range of 2–170 ng/mL for RAM and 8–680 ng/mL for HCTZ. The intra-day and inter-day precisions were <15% and the accuracy was all within ±15% (at LLOQ level ±20%). Additionally, the LC-MS/MS method was fully validated for all the other parameters such as selectivity, matrix effect, recovery and stability as well. In conclusion, the findings of the present study revealed the selectivity and sensitivity of this method for the simultaneous estimation of RAM and HCTZ in human plasma.
Keywords: Bioanalytical method; Carbamazepine; Hydrochlorothiazide; LC-MS/MS; Ramipril;

A new microcolumn-type microchip for examining the expression of chimeric fusion genes using a nucleic acid sandwich hybridization technique by Michihiro Ohnishi; Naoyuki Sasaki; Takuya Kishimoto; Hidetoshi Watanabe; Masatoshi Takagi; Shuki Mizutani; Noriyuki Kishii; Akio Yasuda (60-67).
We report a new type of microcolumn installed in a microchip. The architecture allows use of a nucleic acid sandwich hybridization technique to detect a messenger RNA (mRNA) chain as a target. Data are presented that demonstrate that the expression of a chimeric fusion gene can be detected. The microcolumn was filled with semi-transparent microbeads made of agarose gel that acted as carriers, allowing increased efficiency of the optical detection of fluorescence from the microcolumn. The hybrid between the target trapped on the microbeads and a probe DNA labeled with a fluorescent dye was detected by measuring the intensity of the fluorescence from the microcolumn directly. These results demonstrate an easy and simple method for determining the expression of chimeric fusion genes with no preamplification.
Keywords: Chimeric fusion gene; Gene expression; Nucleic acid sandwich hybridization; Microcolumn chromatography; Semi-transparent microbead; Fluorescent detection;

A high-throughput method for the simultaneous determination of 26 mycotoxins in sesame butter was developed by coupling the modified Quick Easy Cheap Effective Rugged and Safe (QuEChERS) method with ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC–MS/MS). The samples were sequentially extracted using 20 mL (80:20, v/v) and 5 mL (20:80, v/v) acetonitrile aqueous solutions, followed by salting out by the addition of magnesium sulfate and sodium chloride. Finally, the samples were purified using hexane and dispersed C18 solid phase extraction (dSPE). The mycotoxins were further separated using a C18 column and detected by electrospray ionization (ESI) in the multiple reactions monitoring (MRM) mode. Using this detection technique, 16 mycotoxins were detected as positive ions using methanol and water containing 0.1% formic acid as the mobile phase, whereas the other 10 mycotoxins were detected as negative ions using methanol and water as the mobile phase. With the matrix-matched quantification calibration, the developed method showed a good linear dynamic range with regression coefficients of 0.995 or higher. This method allowed for the detection of the 26 mycotoxins at LOQs significantly lower than the available maximum residue levels currently regulated by EU regulations. Additionally, at the three spiking levels examined, the majority of recoveries were within 60–120%, with RSDs within 15%. The method developed herein has the advantages of high sensitivity, accuracy and throughput, and it can be applied to the target screening of mycotoxins in real samples.
Keywords: Mycotoxin; Sesame butter; QuEChERS; UHPLC–MS/MS;

Simultaneous determination of osthole, bergapten and isopimpinellin in rat plasma and tissues by liquid chromatography–tandem mass spectrometry by Jing Li; Bo Ma; Qi Zhang; Xiaojing Yang; Jingjing Sun; Bowen Tang; Guangbo Cui; Di Yao; Lei Liu; Guiying Gu; Jianwei Zhu; Ping Wei; Pingkai Ouyang (77-85).
A highly selective and sensitive method for simultaneous quantitation of osthole, bergapten and isopimpinellin in rat plasma and tissues was developed by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). After liquid–liquid extraction of samples with methyl tert-butyl ether, the analytes and dextrorphan (internal standard, IS) were separated by a Hypersil GOLD AQ C18 column with gradient elution of acetonitrile and water containing 0.5‰ formic acid. Three determinands were detected using an electrospray ionization (ESI) tandem mass spectrometry in the multiple reaction monitoring (MRM) modes with positive electrospray ionization. Calibration curves were recovered over the concentration ranges of 1–200 ng/ml, 1–500 ng/ml, 0.25–200 ng/ml for osthole, bergapten and isopimpinellin in plasma; 1–100 ng/ml, 1–500 ng/ml, 0.5–100 ng/ml for osthole, bergapten and isopimpinellin in tissues, respectively. The intra-day precision (R.S.D.) was within 13.90% and the intra-day accuracy (R.E.) was within −6.27 to 6.84% in all biological matrixes. The inter-day precision (R.S.D.) was less than 13.66% and the inter-day accuracy (R.E.) was within −10.64 to 13.04%. Then the method was successfully applied to investigate plasma pharmacokinetic study and tissue distribution of osthole, bergapten and isopimpinellin in rats after oral administration of Fructus Cnidii extraction, especially for testis/uterus tissue distribution. The results demonstrated that osthole, bergapten and isopimpinellin were absorbed and eliminated rapidly with wide distributions in rats. Distribution data of these three bioactive components in testis/uterus tissues could offer useful information for the further preclinical and clinical studies of Fructus Cnidii in the treatment of genital system disease.
Keywords: Osthole; Bergapten; Isopimpinellin; LC–MS/MS; Pharmacokinetics; Tissue distribution;

The role of temozolomide (TMZ) in treatment of high grade gliomas, melanomas and other malignancies is being defined by the current clinical developmental trials. Temozolomide belongs to the group of alkylating agents and is prescribed to patients suffering from most aggressive forms of brain tumors. The estimation techniques for temozolomide from the extracted plasma or biological samples includes high-performance liquid chromatography with UV detection (HPLC-UV), micellar electrokinetic capillary chromatography (MKEC) and liquid chromatography coupled to mass spectroscopy (LC–MS). These methods suffer from disadvantages like low resolution, low sensitivity, low recovery or cost involvement. An analytical method possessing capacity to estimate low quantities of TMZ in plasma samples with high extraction efficiency (%) and high resolution with cost effectiveness needs to be developed. Cost effective, robust and low plasma component interfering HPLC method using salting out liquid–liquid extraction (SALLE) technique was developed and validated for estimation of drug from plasma samples. The extraction efficiency (%) with conventional LLE technique with methanol, ethyl acetate, dichloromethane and acetonitrile was found to be 5.99 ± 2.45, 45.39 ± 4.56, 46.04 ± 1.14 and 46.23 ± 3.67 respectively. Extraction efficiency (%) improved with SALLE where sodium chloride was used as an electrolyte and was found to be 6.80 ± 5.56, 52.01 ± 3.13, 62.69 ± 2.11 and 69.20 ± 1.18 with methanol, ethyl acetate, dichloromethane and acetonitrile as organic solvent. Upon utilization of two salts for extraction (double salting liquid–liquid extraction) the extraction efficiency (%) was further improved and was twice of LLE. It was found that double salting liquid–liquid extraction technique yielded extraction efficiency (%) of 11.71 ± 5.66, 55.62 ± 3.44, 77.28 ± 2.89 and 87.75 ± 0.89. Hence a method based on double SALLE was developed for quantification of TMZ demonstrating linearity in the range of 0.47–20 μg/ml. The LOQ and LOD for the developed method were 0.4 μg/ml and 0.1 μg/ml, respectively. Thus, plasma non-interfering SALLE-HPLC method that is precise, robust, accurate, specific and cost effective for estimation of temozolomide from plasma samples was developed and validated.
Keywords: HPLC; Temozolomide; SALLE; Salting out; Validation;

Quantitative determination of mithramycin in human plasma by a novel, sensitive ultra-HPLC–MS/MS method for clinical pharmacokinetic application by Jeffrey Roth; Cody J. Peer; Brigitte Widemann; Diane E. Cole; Rachel Ershler; Lee Helman; David Schrump; William D. Figg (95-101).
Mithramycin is a neoplastic antibiotic synthesized by various Streptomyces bacteria. It is under investigation as a chemotherapeutic treatment for a wide variety of cancers. Ongoing and forthcoming clinical trials will require pharmacokinetic analysis of mithramycin in humans, both to see if target concentrations are achieved and to optimize dosing and correlate outcomes (response/toxicity) with pharmacokinetics. Two published methods for mithramycin quantitation exist, but both are immunoassays that lack current bioanalytical standards of selectivity and sensitivity. To provide an upgraded and more widely applicable assay, a UPLC–MS/MS method for quantitation of mithramycin in human plasma was developed. Solid-phase extraction allowed for excellent recoveries (>90%) necessary for high throughput analyses on sensitive instrumentation. However, a ∼55% reduction in analyte signal was observed as a result of plasma matrix effects. Mithramycin and the internal standard chromomycin were separated on a Waters Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and detected using electrospray ionization operated in the negative mode at mass transitions m/z 1083.5   268.9 and 1181.5   269.0, respectively, on an AB Sciex QTrap 5500. The assay range was 0.5–500 ng/mL and proved to be linear (r 2  > 0.996), accurate (≤10% deviation), and precise (CV < 15%). Mithramycin was stable in plasma at room temperature for 24 h, as well as through three freeze–thaw cycles. This method was subsequently used to quantitate mithramycin plasma concentrations from patients enrolled on two clinical trials at the NCI.
Keywords: Mithramycin; Ultra-HPLC; Tandem mass spectrometry;

Enhancement of specificity of aldosterone measurement in human serum and plasma using 2D-LC–MS/MS and comparison with commercial immunoassays by Julie A. Ray; Mark M. Kushnir; John Palmer; Seyed Sadjadi; Alan L. Rockwood; A. Wayne Meikle (102-107).
Accurate measurement of aldosterone is important for the standardized testing of primary hyperaldosteronism. Commercial immunoassays show substantial between-method variations resulting in significant clinical consequences. We developed a specific two dimensional (2D)-LC–MS/MS method for measuring aldosterone in human serum and plasma and compared it with three commercial immunoassays and an LC–MS/MS method.250 μL samples, controls and calibrators spiked with d4-aldosterone were subjected to liquid–liquid extraction. The samples were analyzed using negative mode electrospray and 2D-LC followed by MS detection using an ABSciex 5500 mass spectrometer and compared with immunoassays of Siemens (Coat-A-Count), DiaSorin (CLIA-LIAISON), and IBL (ELISA). Data was acquired using multiple reaction-monitoring mode.LOQ and LOD of the method were 0.04 and 0.02 nmol/L respectively. The assay was linear up to 166 nmol/L. Inter and intra-assay imprecision at 0.13, 1.38 and 8.30 nmol/L were <10%. Interferences were absent and no differences were observed between serum and plasma matrices. Method recovery ranged from 95% to 113%. Ion suppression was not observed. Evaluated immunoassays showed positive biases ranging between 22% and 37% when compared with the developed method.We developed and validated an accurate method for measurement of aldosterone in human serum and plasma using 2D-LC–MS/MS which is suitable for clinical purposes. The method is faster than previously published LC–MS/MS methods, uses less sample, has adequate sensitivity while being able to preserve high specificity in a cost effective manner. Linearity of the assay makes it promising for urine and adrenal venous samples. Comparison with three commercial immunoassays demonstrates the advantages of the developed method.
Keywords: 2D-LC–MS/MS; Aldosterone; Hypertension; Immunoassay; Liquid–liquid extraction; SLE;

A method incorporating HPLC-PDA-IT-MS n with HPLC-Quadrupole-Orbitrap-MS was developed for the investigation of chemical fingerprint of Citrus reticulate ‘Chachi’ decoction (CRCD) and metabolic profile of SD rat plasma sample after oral administration of CRCD (1.5 g herb/kg). A total of 27 chemical constituents of CRCD were identified from their MW, UV spectra, MS n data and retention behavior by comparing the results with those of the reference standards or literature. And 43 compounds were detected in dosed SD rat plasma samples, including 9 prototypes which were identified as hesperetin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein, nobiletin, tetramethyl-O-scutellarein, HMF (3,5,6,7,8,3′,4′-heptamethoxyflavone), tangeretin and 5-demethylnobiletin and 34 metabolites underwent metabolic process of demethylation, glucuronide conjugation, sulfate conjugation or mixed modes. This is the first research for the metabolic profile of CRCD in SD rats, which could lay a foundation for the further studies of CRC or its formulation.
Keywords: HPLC-PDA-IT-MS n ; HPLC-Quadrupole-Orbitrap-MS; Chemical fingerprint; Metabolic profile; Citrus reticulate ‘Chachi’ decoction;

Method for the quantification of current use and persistent pesticides in cow milk, human milk and baby formula using gas chromatography tandem mass spectrometry by Xianyu Chen; Parinya Panuwet; Ronald E. Hunter; Anne M. Riederer; Geneva C. Bernoudy; Dana Boyd Barr; P. Barry Ryan (121-130).
The aim of this study was to develop an analytical method for the quantification of organochlorine (OC), organophosphate (OP), carbamate, and pyrethroid insecticide residues in cow milk, human milk, and baby formula. A total of 25 compounds were included in this method. Sample extraction procedures combined liquid–liquid extraction, freezing-lipid filtration, dispersive primary–secondary amine cleanup, and solid-phase extraction together for effective extraction and elimination of matrix interferences. Target compounds were analyzed using gas chromatography with electron impact ionization-tandem mass spectrometry (GC-EI-MS/MS) in the multiple reaction monitoring (MRM) mode. Average extraction recoveries obtained from cow milk samples fortified at two different concentrations (10 ng/mL and 25 ng/mL), ranged from 34% to 102%, with recoveries for the majority of target compounds falling between 60% and 80%. Similar ranges were found for formula fortified at 25 ng/mL. The estimated limits of detection for most target analytes were in the low pg/mL level (range 3–1600 pg/mL). The accuracies and precisions were within the range of 80–120% and less than 15%, respectively. This method was tested for its viability by analyzing 10 human milk samples collected from anonymous donors, 10 cow milk samples and 10 baby formula samples purchased from local grocery stores in the United States. Hexachlorobenzene, p,p-dicofol, o,p-DDE, p,p-DDE, and chlorpyrifos were found in all samples analyzed. We found detectable levels of permethrin, cyfluthrin, and fenvalerate in some of the cow milk samples but not in human milk or baby formula samples. Some of the pesticides, such as azinphos-methyl, heptachlor epoxide, and the pesticide synergist piperonyl butoxide, were detected in some of the cow milk and human milk samples but not in baby formula samples.
Keywords: Current use and persistent pesticides; Whole milk; Human milk; Baby formula; Tandem mass spectrometry; Gas chromatography;