HPLC-UV assay for monitoring total and unbound mycophenolic acid concentrations in children

A simple, accurate and sensitive HPLC method was developed for measuring total and unbound mycophenolic acid (MPA) in human plasma. Total MPA was extracted by protein precipitation and ultrafiltration was used to assess unbound MPA concentrations. The supernatant (20 microL) or ultrafiltrate (100 microL) was injected onto a C(18) HPLC column with a mobile phase of 0.05 m sodium phosphate buffer (pH 2.31)-acetonitrile (55:45, v/v for total MPA; 50:50 for unbound MPA) with UV detection at 254 nm. The extraction recovery was over 93% and reproducible. The assay was linear over the concentration range of 0.07-50 mg/L for total MPA and 4-1500 microg/L for unbound MPA. Intra- and inter-day assay reproducibility was less than 10%. Detection limits were 0.04 mg/L and 2 microg/L for total and unbound MPA, respectively. The assay utility was established in samples collected from five paediatric bone marrow transplant recipients who were receiving intravenous doses of mycophenolate mofetil. In these patients MPA concentrations ranged from 0.07 to 7.83 mg/L and unbound drug concentrations ranged from 2.1 to 107.5 microg/L. This method can be effectively applied to MPA pharmacokinetics in paediatric patients.     

Monitoring volatile compounds during dry-cured ham ripening by solid-phase microextraction coupled to a new direct-extraction device

Key flavour volatile compounds were monitored during ripening of dry-cured ham by solid-phase microextraction (SPME) coupled to a new direct-extraction device (DED). DED allows the insertion of the SPME fibre into the core of solid materials with no damage to the fibre. This enables extraction of volatiles from solid foodstuffs while avoiding sample handling. Major groups of volatile compounds extracted with SPME-DED agreed with available scientific literature about dry-cured ham volatiles. Moreover, volatile compounds previously highlighted as quality markers in dry-cured ham, such as 3-methylbutanal or hexanal, were satisfactorily extracted using SPME-DED. Changes in the profile of volatile compounds throughout the processing followed a typical pattern of volatile compounds formation. Therefore, SPME-DED appears as a new and promising method for monitoring ripening of dry-cured hams with no depreciation of the product, which might substitute traditional subjective methods currently used in the ham processing industry. However, the use of the internal standard method is not possible with this technique. Therefore, results using SPME-DED only point out a trend in the volatile profile. Further attempts relating data obtained using SPME-DED in dry-cured hams with sensory and chemical data from the same samples would be necessary for optimising this method as a quality control method in dry-cured ham industries.     

Determination of biogenic amines in fresh and processed meat by suppressed ion chromatography-mass spectrometry using a cation-exchange column

A new method for simultaneous determination of underivatized biogenic amines based on the separation by cation-exchange chromatography and suppressed conductivity coupled with mass spectrometry detection has been developed. The method has been applied to the analysis of cadaverine, putrescine, histamine, agmatine, phenethylamine and spermidine in processed meat products. The amines were extracted from muscle tissue with methanesulfonic acid without any additional derivative step or sample clean-up. Biogenic amines were separated by the IonPac CS17 column, a cation-exchange column used with gradient elution, and detection was done by suppressed conductivity and mass spectrometry. Tyramine was simultaneously analysed by using a spectrophotometer (275 nm) before the suppressed conductivity detection. Linearity of response was obtained in the range 0.25-25 microg mL(-1). The detection limits ranged from 23 microg L(-1) for putrescine to 155 microg L(-1) for spermidine (suppressed conductivity) and from 9 microg L(-1) for agmatine to 34 microg L(-1) for spermidine (MS). Average recoveries from meat samples ranged from 85 to 97% and coefficients of variation ranged from 4.5 to 9.7%. The analysis of biogenic amines in fresh and processed meats (dry-cured, cooked and fermented products) can be used as a quality marker of raw material and for studying the relationship between their changes and the fermentation process involved in dry sausage ripening.     

Determination of gestagens in kidney fat by liquid chromatography tandem mass spectrometry

The use of gestagens in animal fattening is prohibited within the European Union. Recently, the use of spectrometric methods for the detection and confirmation of banned substances was made obligatory. Therefore, conventional high-performance liquid chromatographic (HPLC) methods have been superseded. It has been possible to couple a previously described HPLC method for the determination of acetyl-gestagens in kidney fat to tandem mass spectrometry (LC-MS/MS). The decision limits CCalpha and the detection capability CCbeta are found to be below the minimum required performance limit (MRPL) established for medroxyprogesterone acetate (MPA) at 1 microg kg(-1). The calculated values for CCalpha are as follows: megestrol acetate (MGA)--0.15 microg kg(-1), melengesterol acetate (MLA)--0.15 microg kg(-1), chlormadinone acetate (CMA)--0.37 microg kg(-1) and for medroxyprogesterone acetate (MPA)--0.24 microg kg(-1). The CCbeta values for these compounds have been determined as 0.19, 0.19, 0.47 and 0.32 microg kg(-1), respectively.     

Simultaneous determination of mycophenolic acid and valproic acid based on derivatization by high-performance liquid chromatography with fluorescence detection

A reliable and validated reversed-phase high-performance liquid chromatography (HPLC) method using fluorescence detection is reported for the simultaneous quantitation of mycophenolic acid (MPA) and valproic acid (VPA) in human plasma. The method is based on the pre-column derivatization of valproic acid with 4-bromomethyl-6, 7-dimethoxycoumarin (BrMMC) and online solvatochromism of MPA by pH adjustment. The linear calibration range was 0.50-30 microg/mL for MPA and 5.00-150 microg/mL for VPA. The relative standard deviations of the method of intra- and inter-day analyses (n = 6) were below 6.5 and 6.7% for MPA, and 5.8 and 6.3% for VPA, respectively. Dichloromethane was used for the simultaneous extraction of MPA and VPA from acidified plasma. This reliable method can be applied in the analysis of MPA and VPA in human plasma using only a small volume (100 microL).     

Determination of mycophenolic acid in human plasma by ultra‐performance liquid chromatography–tandem mass spectrometry and its pharmacokinetic application in kidney transplant patients

To implement and validate an analytical method by ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC MS/MS) to quantify mycophenolic acid (MPA) in kidney transplant patients. Quantification of MPA was performed in an ACQUITY UPLC H Class system coupled to a Xevo TQD detector and it was extracted from plasma samples by protein precipitation. The chromatographic separation was achieved through an ACQUITY HSS C₁₈ SB column with 0.1% formic acid and acetonitrile (60:40 vol/vol) as mobile phase. The pharmacokinetic parameters were calculated by non‐compartmental analysis of MPA plasma concentrations from 10 kidney transplant patients. The linear range for MPA quantification was 0.2–30 mg/L with a limit of detection of 0.07 mg/L; the mean extraction recovery was 99.99%. The mean intra‐ and inter‐day variability were 2.98% and 3.4% with a percentage of deviation of 8.4% and 6.6%, respectively. Mean maximal concentration of 10 mg/L at 1.5 h, area under the concentration–time curve of 36.8 mg·h/L, elimination half‐life of 3.9 h, clearance of 0.32 L/h/kg and volume of distribution of 1.65 L/kg were obtained from MPA pharmacokinetics profiles. A simple, fast and reliable UPLC–MS/MS method to quantify MPA in plasma was validated and has been applied for pharmacokinetic analysis in kidney transplant patients.     

Determination of beta-agonists in liver and retina by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of bromobuterol, cimaterol, clenbuterol, clenpenterol, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritrodrine, salbutamol, terbutaline, and tulobuterol residues in bovine liver and retina is reported. This procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on Oasis HLB solid-phase extraction cartridges, followed by determination of the residues by LC-tandem quadrupole MS using atmospheric pressure chemical ionization in the positive ion mode. Overall average recoveries ranged from 23 to 76% for liver and 34 to 77% for retina. The mean values for samples fortified at levels between 0.5-2.0 microg/kg (liver) and 5-20 microg/kg (retina) agreed within 98-118% of the spiked levels, with coefficients of variation ranging from 6 to 20%. The decision limits, CCalpha, ranged from 0.1 to 0.3 microg/kg for liver, 1-3 microg/kg for retina, and detection capabilities, CCbeta, from 0.2-0.5 microg/kg for liver and 2-5 microg/kg for retina.     

液相色谱-串联质谱法测定肉及肉制品中利谷隆及其代谢产物3,4-二氯苯胺残留

建立了液相色谱-串联质谱分析多种肉及肉制品中利谷隆及其代谢产物3,4-二氯苯胺残留的方法。样品用丙酮-乙腈(5:95, v/v)提取,冷冻去脂,经弗罗里硅土固相萃取柱净化后进行液相色谱-串联质谱检测,采用内标法定量。利谷隆及3,4-二氯苯胺在1～500 μg/L范围内呈良好的线性关系,相关系数(r)均大于0.998,方法的定量限(信噪比(S/N)＞10)为10 μg/kg,检出限(S/N＞3)为5 μg/kg。在各种肉及肉制品基质中添加低、中、高3种不同水平的利谷隆及3,4-二氯苯胺,其回收率范围分别为88.3%～101.2%和91.6%～101.6%,相对标准偏差(RSD)分别为4.8%～13.7%和4.7%～11.8%。结果表明所建立的方法可以满足肉及肉制品中利谷隆和3,4-二氯苯胺残留量的检测需要。     

Catalytic thermometric titrations in non-aqueous solvents by coulometrically generated titrant

Catalytic thermometric titrations have been developed for tertiary amines and salts of organic acids in acetic and propionic anhydride with titrant coulometrically generated at a mercury and/or platinum anode, hydroquinone being added to the solution titrated if the platinum anode is used. The results obtained are compared with those obtained by coulometric titration with the end-point detected either photometrically or potentiometrically. On a élaboré des titrages thermométriques catalytiques pour les amines tertiaires et les sels d'acides organiques en anhydrides aétique et propionique avec l'agent de titrage engendré coulométriquement sur une anode de mercure et/ou platine, de l'hydroquinone étant ajoutée à la solution titrée si l'on emploie l'anode de platine. Les résultats obtenus sont comparés avec ceux obtenus par titrage coulométrique avec le point de fin de réaction détecté soit photométriquement soit potentioétriquement.     

Simultaneous neutron-activation determination of selenium and mercury in biological samples by volatilization

A method is described for the determination of selenium together with mercury in biological samples by neutron-activation analysis based on quantitative volatilization of both elements. The technique originally developed for mercury, based on pyrolysis with filtration of undesirable impurities and selective trapping from the gas phase, is now extended to selenium. The radionuclides (197)Hg and (75)Se, from one sample, are trapped separately and counted in a well-type NaI(Tl) detector and gamma-spectrometer for maximum sensitivity. The method has been tested by comparative analyses and analyses of standard biological materials, and gives good results. It is simple and is especially effective in studies of the interaction of mercury and selenium in biological systems; a positive correlation for these elements was found for human tissues. On décrit une méthode pour le dosage du sélénium conjointement au mercure dans les échantillons biologiques par analyse par activation de neutrons basée sur la volatilisation quantitative des deux éléments. La techniqu initialement développée pour le mercure, basée sur la pyrolyse avec filtration des impuretés indésirables et captage sélectif de la phase gazeuse, est maintenant étendue au sélénium. Les radionuclides (197)Hg et (75)Se, d'un échantillon, sont captés séparément dans un détecteur NaI(Tl) du type puits et un spectromètre gamma pour la sensibilité maximale. La méthode a été essayée par des analyses comparatives et des analyses de produits biologiques étalons, et donne de bons résultats. Elle est simple et particulièrement efficace dans les études de l'interaction du mercure et du sélénium dans des systèmes biologiques; on a trouvé une corrélation positive pour ces éléments pour des tissus humains.     

Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with chloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one pork sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.     

反相高效液相色谱法同时测定4种氟喹诺酮类药物在鸡可食性组织中的残留

建立了一种可同时测定鸡可食性组织中环丙沙星、达氟沙星、恩诺沙星及沙拉沙星等多种残留的反相高效液相色谱 -荧光分析法。鸡的肌肉、皮和脂、肝、肾等4种组织经不同pH值的磷酸二氢钾缓冲溶液匀浆提取,上清液通过C18固相萃 取柱净化,以流动相洗脱。洗脱液经液相色谱分离后,用荧光检测器进行检测(激发波长280 nm, 发射波长450 nm),外标 法定量。对鸡的4种组织进行添加回收率测定,结果显示方法在添加水平为20~300 μg/kg时药物的回收率约为53.9%～93.4%,批间回收率测定值的相对标准偏差低于23%;环丙沙星、恩诺沙星、沙拉沙星 的定量检出限为20 μg/kg,达氟沙星为4μg/kg。方法简单、快速,能满足常规兽药残留检测的需要。     

Determination of the Fusarium mycotoxins, fusaproliferin and beauvericin by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A method is described using LC-MS for the detection of the mycotoxins fusaproliferin (FUS) and beauvericin (BEA) in cultures of Fusarium subglutinans and in naturally contaminated maize. Protonated molecular ion signals for FUS and BEA were observed at m/z 445 and m/z 784, respectively. Collision induced dissociation of the readily dehydrated protonated molecular ion of the sesterterpene FUS (m/z 427) led to the loss of another water molecule (m/z 409) and acetic acid (m/z 385), while the cyclic lactone trimer BEA fragmented to yield the protonated dimer (m/z 523) and monomer (m/z 262), respectively. Detection of FUS was best performed in the MS-MS mode while BEA displayed a stronger signal in the MS mode. The on-column instrumental detection limits for pure FUS and BEA were found to be 2 ng and 20 pg (S/N=2) while those in naturally contaminated maize were 1 microg/kg and 0.5 microg/kg, respectively. Five South African strains of F. subglutinans were analyzed following methanol extraction of which four produced FUS at levels between 330 mg/kg and 2630 mg/kg while only three produced BEA at levels between 140 mg/kg and 700 mg/kg. Application of this method to naturally contaminated maize samples from the Transkei region of South Africa showed FUS at levels of 8.8-39.6 microg/kg and BEA at 7.6-238.8 microg/kg.     

Quantification of residual nitrite and nitrate in ham by reverse-phase high performance liquid chromatography/diode array detector

Nitrite and nitrate are used as additives in ham industry to provide colour, taste and protect against clostridia. The classical colorimetric methods widely used to determine nitrite and nitrate are laborious, suffer from matrix interferences and involve the use of toxic cadmium. The use of chromatography is potentially attractive since it is more rapid, sensitive, selective and provides reliable and accurate results. A rapid and cost-effective RP-HPLC method with diode array detector was optimized and validated for quantification of nitrites and nitrates in ham. The chromatographic separation was achieved using a HyPurity C18, 5 μm chromatographic column and gradient elution with 0.01 M n-octylamine and 5 mM tetrabutylammonium hydrogenosulphate to pH 6.5. The determinations were performed in the linear range of 0.0125–10.0 mg/L for nitrite and 0.0300–12.5 g/L for nitrate. The detection limits were 0.019 and 0.050 mg/kg, respectively. The reliability of the method in terms of precision and accuracy was evaluated. Coefficients of variation lower than 2.89% and 5.47% were obtained for nitrite and nitrate, respectively (n = 6). Recoveries of residual nitrite/nitrate ranged between 93.6% and 104.3%. Analysis of cooked and dried ham samples was performed, and the results obtained were in agreement with reference procedures.     

Simultaneous determination of 20 beta-agonists in pig muscle and liver by high-performance liquid chromatography/tandem mass spectrometry

A method was developed to determine 20 illegal residual beta-agonists in pork tissues, including muscle and liver simultaneously. The samples were hydrolyzed by beta-glucuronidase, purified by PCX SPE cartridges, and detected by HPLC coupled with electrospray ionization MS/MS operating in the positive ion mode. Matrix-fortified calibration was performed to compensate for the matrix effect and loss in sample preparation. Decision limit ranged from 0.05 to 0.23 microg/kg in muscle and 0.05 to 0.57 microg/kg in liver. Decision capacity ranged from 0.11 to 0.4 microg/kg in muscle and 0.16 to 0.79 microg/kg in liver. In Food Analysis Performance Assessment Scheme proficiency test 0287, a pig liver test material containing 13 beta-agonists was analyzed using the method developed, and clenbuterol and ractopamine were confirmed as being present. Z-scores for clenbuterol and ractopamine were 0.2 and 0.6, respectively.     

Determination of acrylamide in potato crisps by capillary electrophoresis with quantum dot-mediated LIF detection

Acrylamide or 2-propenamide (AAM), a water-soluble toxic contaminant, has recently caused health concern after it was found in food products made by high-temperature cooking. Due to its weak UV absorption and electrochemically inactive state, common analytical methods do not have sufficient sensitivity to meet the World Health Organization requirement. A LIF detection method mediated by water-soluble CdTe quantum dots capped with mercaptopropyl acid (MPA) is thus developed for AAM quantitation. The optimized conditions are as follows: 30 mmol/L SDS, 0.1 mmol/L quantum dot, and 40 mmol/L phosphate buffer solution at pH 8.0 under 18 kV run voltage with LIF detection at 473 nm excitation and 568 nm fluorescence. The linear quantitation range for AAM was found to vary from 1.0 to 100 mg/kg and a detection limit (S/N=2) at 0.1 mg/kg, showing sufficient sensitivity to meet the maximum AAM specified by the Joint Food and Agriculture Organization/World Health Organization Expert Committee for potato crisps. Recoveries for potato crisps sample spiked with 10, 20, and 100 mg/kg AAM were found to vary between 90 and 95% with RSD <5.7% (n=3).     

气相色谱-质谱法测定动物组织中三种1,2-二苯乙烯类药物的残留量

建立了动物组织中己烯雌酚(DES)、己二烯雌酚(DIS)和己烷雌酚(HS)残留量的气相色谱-质谱分析方法。动物组织样品在碱性条件下用乙酸乙酯提取,经碳酸钠溶液液-液分配净化,再用硅胶柱固相萃取净化,洗脱液均分成两份后经氮气吹干,分别用双三甲基硅基三氟乙酰氨(BSTFA)和七氟丁酸酐(HFBA)衍生,采用选择离子监测模式(SIM)进行测定,外标法定量。检出限为0.30 μg/kg(cis-DES),0.10 μg/kg(trans-DES和HS)和0.15 μg/kg(DIS)。在0.5~4.0 μg/kg添加水平,回收率为73.0%～86.5%,相对标准偏差(RSD)为1.0%～7.2%。衍生物的峰面积与样品浓度在10～1000 μg/L(DES和DIS) 和5～500 μg/L(HS)范围内呈良好的线性关系,线性回归系数大于0.99。     

Rapid method for the determination of non-steroidal anti-inflammatory drugs in animal tissue by liquid chromatography-mass spectrometry with ion-trap detector

A rapid and new liquid chromatography-mass spectrometry with ion-trap detection method for the determination of meloxicam (MLX), flunixin meglumine (FLU), carprofen (CPF), and tolfenamic acid (TOLF) in animal tissue is described. MRLs between 10 and 500 microg kg(-1) in muscle and between 65 and 1000 microg kg(-1) in liver, from different animal species have been established in the EU for these compounds. After chemical hydrolysis, an organic extraction from homogenised tissue was performed. Final extract was injected in a liquid chromatograph with an ion-trap mass spectrometer with electrospray interface. Four identification points (one precursor and two product ions) and a minimum of one ion ratio was monitored for each compound. For quantitative purposes flunixin-D3 (FLU-D3) was used as internal standard. The method was validated using fortified blank muscle and liver from different animal species according to the 2002/657/EC European decision criteria. The decision limits (CCalpha) and detection capabilities (CCbeta) were determined and their values were at concentrations near the MRL for each substance.     

Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection. Optimization and validation

A high-performance liquid chromatography-diode array detection method (HPLC-DAD) combined with liquid chromatography-mass spectrometry was developed for the determination of enrofloxacin and its metabolite ciprofloxacin in goat milk. The HPLC-DAD method validation was compliant with the "DG SANCO 1805/2000" European regulation. The residues were extracted from milk with phosphate buffer, purified on a C18 Speedisk cartridge SPE (Baker) and then analysed using HPLC-DAD set at 277 nm. The decision limit (CCa) calculated by spiking samples at 100 microg/kg with both analytes, taking into account the maximum residue limit (MRL) of 100 microg/kg established by the European Union for the sum of enrofloxacin and its metabolite ciprofloxacin in milk, was 105.3 microg/kg for enrofloxacin and 105.5 microg/kg for ciprofloxacin. The detection capability (CCbeta) was 110.7 and 110.9 microg/kg for enrofloxacin and ciprofloxacin, respectively. The mean recoveries of the method, calculated by spiking samples at 50, 100 and 150 microg/kg were 84% for enrofloxacin and 88% for ciprofloxacin. The limit of quantification was 20 microg/kg for both analytes. The HPLC-DAD validated method was successfully applied for the first time in goats milk, and proved to be suitable for the sensitive and accurate quantification and confirmation analysis of enrofloxacin and ciprofloxacin for regulatory purposes.     

Simultaneous determination of betamethasone and dexamethasone residues in bovine liver by liquid chromatography/tandem mass spectrometry

In this study a new method for the simultaneous confirmation of betamethasone and dexamethasone residues in bovine liver is presented. A Quattro LCZ triple quadrupole mass spectrometer, equipped with an atmospheric pressure ionization (API) source, was coupled to a high performance liquid chromatograph (HPLC) system. Spiked liver samples were first extracted with acetonitrile, and the extracts were purified on C-18 columns. LC separations were performed on a Hypercarb column, with acetonitrile/water (90:10, v/v, +0.3% formic acid) as the mobile phase. Retention times for dexa- and betamethasone were 6.60 and 8.50 min, respectively. Fluorometholone had a retention time of 6.70 min and was used as the internal standard. The detection of the analytes was performed in the multiple reaction monitoring (MRM) mode. The assay was linear over the range of 0.5 to 8 microg/kg for both analytes. The estimated determination limits were 0.2 microg/kg for both beta- and dexamethasone and the quantification limits were 0.4 microg/kg for dexamethasone and 0.3 microg/kg for betamethasone. Analysis precision at 1, 2 and 4 microg/kg was lower than 6.1% (relative standard deviation, RSD) and accuracy was at least 97.5%. Recoveries at 1, 2 and 4 microg/kg ranged between 56 and 69%.