A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients.We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA),satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.     

Routine method for the simultaneous quantification of 17alpha-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry

Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic-mass spectrometric method for the simultaneous quantification of 17alpha-hydroxyprogesterone (17alphaOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Delta4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5 mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1 ng/mL. The limit of quantification was 0.1 ng/mL for all steroids except cortisol which was at 0.25 ng/mL. d3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10 ng/mL (n = 10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10 ng/mL and ranged from -9.2% to 10.6% (n = 10). Linear calibration equations were obtained for all five steroids (0.125-31.25 ng/mL) and for cortisol (0.125-200 ng/mL). Relative recoveries at concentrations 1.0 and 12.5 ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.     

Determination of dimethoate and omethoate in human serum samples. Risk assessment for the operator

A simple and effective analytical procedure has been developed for the determination of dimethoate (DIM) residues and its metabolite, omethoate, in serum samples of pesticide operators. For the selection of the most appropriate method for sample treatment, techniques such as headspace solid phase micro extraction and solid phase extraction and liquid–liquid extraction were applied. The applied method was based on toluene (2?mL) extraction of a 0.5?mL serum sample. In this report, it was observed that DIM concentration level affected the ratio of the area response of DIM and one of its oxygenated metabolite, omethoate. In this context, higher concentrations favoured the predominance of DIM while lower concentrations lead to the formation of omethoate. The method was validated using human serum samples spiked with DIM. Good linearity was obtained in the range of 1–10?ng/mL co-calculating DIM and omethoate. Various concentrations of DIM were mixed with serum and stored up to five days at ?20°C. Recoveries ranged from 72% to 88% at two spiking levels for six replicates. The detection and quantification limit were calculated at 0.12 and 0.36?ng/mL of serum, respectively. Finally the comparison with the Acceptable Operator Exposure Level (AOEL) of DIM revealed that the maximum exposure of the operators reached the 30% of the AOEL for only two cases.     

Nickel quantification in serum by a validated sector-field inductively coupled plasma mass spectrometry method: Assessment of tentative reference values for an Italian population

The daily exposure to Ni from food, industrial processes, jewellery and coins makes the determination of Ni in human serum an important way to monitor the health status in non-occupationally exposed subjects. To this end, a method based on sector-field inductively coupled plasma mass spectrometry was developed and validated. The limits of detection (LoD) and quantification (LoQ), sensitivity, linearity range, trueness, repeatability, within-laboratory reproducibility and robustness were the considered issues of the validation process. The uncertainty associated with the measurements was also calculated, according to the Eurachem/Citac Guide. The method LoD and LoQ were 0.03 and 0.09 ng mL(-1), linearity was over two order of magnitude, trueness was -3.57%, and the repeatability and reproducibility showed relative standard deviations equal to 4.56% and 6.52%, respectively. The relative expanded uncertainty was 21.8% at the Ni levels found in the general population. The tentative reference value for serum Ni was 0.466 +/- 0.160 ng mL(-1) with a related interval between 0.226 and 1.026 ng mL(-1).     

基于氧化镍沉积硅胶固相萃取与液相色谱-质谱联用技术的2型糖尿病血清中咪唑丙酸的检测

咪唑丙酸可以通过哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)路径引起人的胰岛素抵抗, 从而导致2型糖尿病(T2D), 因此, 咪唑丙酸的准确定量可辅助2型糖尿病的诊断. 本文利用NiO与咪唑基团之间的配位作用, 采用氧化镍沉积硅胶(NiO@SiO₂)萃取材料对咪唑丙酸进行选择性富集和萃取. 首先对NiO@SiO₂ 固相萃取(SPE)条件进行优化: 吸附剂用量为200 mg, 上样液为20 mmol/L的磷酸盐缓冲溶液(pH=3.0), 解吸液为1.0 mL含1%(质量分数)NH₃·H₂O的水溶液; 然后, 对萃取液进行高效液相色谱-质谱(HPLC-MS)分离分析, 建立了血清中咪唑丙酸的检测方法. 结果表明, 咪唑丙酸浓度在0.05~10 ng/mL范围内对质谱响应值具有良好的线性关系(R ²≥0.996), 检出限和定量限分别为0.02和0.05 ng/mL, 加标回收率为84.0%~119%, 相对标准偏差RSD<17.2%. 将该方法用于检测2型糖尿病患者与正常人血清样品中的咪唑丙酸, 发现在2型糖尿病患者与正常人的血清中咪唑丙酸含量存在显著性差异, 说明咪唑丙酸的准确定量对2型糖尿病具有医学诊断上的潜力.     

Determination of artemisinin in human serum by high-performance liquid chromatography with on-line UV irradiation and peroxyoxalate chemiluminescence detection

Artemisinin is an antimalarial drug containing an internal endoperoxide linkage in its structure. A simple, selective and sensitive high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence (PO-CL) method for the determination of artemisinin was developed. This method is based on the fact that endoperoxide in artemisinin structure can be converted to hydrogen peroxide (H(2)O(2)) under ultraviolet (UV) irradiation and the generated hydrogen peroxide can be measured using PO-CL detection. The HPLC-PO-CL system was optimized on a mobile phase, post column chemiluminescence reagent, UV source and irradiation time. In addition, the system was combined with simple liquid-liquid extraction using n-hexane that allowed selective and sensitive determination of artemisinin in serum. The limit of detection using 0.5 mL of blood was 0.062 micromol/L (17.5 ng/mL) at a signal-to-noise ratio of 3. Calibration curve obtained for artemisinin in human serum 4-80 micromol/L (1.1-22.6 microg/mL) showed a good linearity (r = 0.999).     

液相色谱-串联质谱法同时测定动物血浆中21种霉菌毒素或其代谢物残留

建立了同时检测动物血浆中黄曲霉毒素B1等21种霉菌毒素或其代谢物残留的液相色谱-串联质谱方法.动物血浆样品中加入0.1％甲酸-乙腈溶液、NaCl和无水MgSO4进行萃取,无水MgSO4和C18,PSA,A-AL对提取液进行脱水净化,经浓缩、复溶和离心后,再进行测定.采用反相C18色谱柱分离,以0.1％甲酸-0.5 mmol/L乙酸铵溶液和0.1％甲酸-甲醇溶液作为流动相进行梯度洗脱,采用电喷雾离子源(ESI)多反应监测离子模式(MRM)进行检测,基质标准曲线外标法进行定量分析,线性范围在0.05 ～ 100 ng/mL之间,方法的定量限为0.05 ～0.5 ng/mL.在高、中、低3个添加浓度水平下,21种霉菌毒素的平均回收率为62.0％ ～ 116.4％,相对标准偏差小于19％.     

Micellar electrokinetic chromatography with laser-induced fluorescence detection for sensitive determination of ephedrine and pseudoephedrine

A selective and sensitive micellar electrokinetic chromatography method with laser-induced fluorescence detection was developed for the quantification of ephedrine (E) and pseudoephedrine (PE) derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. After conducting a series of optimizations, a running buffer of 10 mM sodium borate + 16 mM SDS was used for separation of the derivatives. A linear relationship for E and PE was obtained in the range of 0.044-6.6 microg mL(-1) (correlation coefficient: 0.9943 for E, 0.9946 for PE), and the detection limits for E and PE were 0.70 and 0.30 ng mL(-1), respectively. The sensitivity of E and PE was improved by several multiples of ten over those of CZE-LIF method. The method was applied to the analysis of the two alkaloids in ephedra herbal medicine and preparations with recoveries in the range of 98.3-107.1%.     

GC-FID/MS method for the impurity profiling of synthetic d-allethrin

A GC/FID/MS method was developed for the identification and quantification of d-allethrin (DA) and its major impurities in commercial samples. Optimisation of the experimental conditions was carried out considering such important requirements as resolution, reproducibility, detection limits of 0.1% (m/m) for the impurities, and short analysis time. Under the optimised final conditions the method was validated for specificity, precision (CV% = 0.133 at 2.10 mg/mL and CV% = 0.035 at 3.00 mg/mL), linearity (0-3.00 microg injected), limits of detection (0.09 ng injected) and quantitation (0.28 ng injected), and robustness. The DA related impurities were identified by using a GC/MS method with ion trap mass detection and also by comparison with synthesised standards. The most abundant impurities were crysolactone, allethrolone, chrysanthemic acid, and chloro-derivatives of DA.     

Method for simultaneous analysis of eight vitamin E isomers in various foods by high performance liquid chromatography and fluorescence detection

The objective of this study was to optimize a method to investigate the occurrence and to quantify the full isomeric composition of vitamin E (α-, β-, γ- and δ-tocopherols and tocotrienols) in 6 vegetables (raw and cooked), 3 herbs/spices, raw and cooked eggs, vegetable oils (canola, olive and soybean), flaxseed and sorghum (flour and seeds) and soy (flour) by HPLC with fluorescence detection. Different conditions of extraction and analysis were tested. The optimized method consisted of direct extraction with solvent (hexane:ethyl acetate, 85:15, v/v). For analysis normal phase column was used with mobile phase consisting of hexane:isopropanol:acetic acid (98.9:0.6:0.5) with isocratic elution and fluorescence detection. Excellent separation of all isomers was obtained along with adequate quantification in the foods analyzed. Recovery rates of standards ranged from 91.3 to 99.4%. The linearity range for each isomer varied from 2.5 to 137.5 ng/mL (R2 greater than 0.995 in all cases). Detection limits ranged from 21.0 to 48.0 ng/mL for tocopherols and from 56.0 to 67.0 ng/mL for tocotrienols, while quantification limits ranged from 105.0 to 240.0 ng/mL for tocopherols and from 280.0 to 335.0 ng/mL for tocotrienols. The optimized method was considered simple, fast and reliable, and also preserved vitamin E isomers when compared to validated methods involving saponification.     

Semi-micro column high-performance liquid chromatography with UV detection for quantification of aspirin and salicylic acid and its application to patients' sera administered with low-dose enteric-coated aspirin

A simultaneous determination of aspirin (ASA) and its metabolite, salicylic acid (SA), in human serum by a semi-micro column HPLC-UV was developed. A relatively small size of serum sample (100 microL) containing ASA and SA was cleaned up by a simple solid phase extraction. A good separation of ASA and SA could be achieved within 25 min using a semi-micro ODS column with an eluent of MeOH/0.7 mm phosphoric acid solution (pH 2.5) = 50:50 (v/v). The calibration curves for ASA and SA showed good linearity (r = 0.999) with the detection limits 114 and 38 ng/mL at a signal-to-noise ratio of 3, respectively. ASA and SA in patients' sera administered with low-dose enteric-coated aspirin were determined, and the concentration ranges obtained for ASA and SA were 1.2-2.2 and 0.5-57.3 microg/mL, respectively.     

Quantification of controlled release leuprolide and triptorelin in rabbit plasma using electromembrane extraction coupled with HPLC–UV

An electromembrane extraction followed by HPLC–UV technique was developed and validated for quantification of leuprolide and triptorelin in rabbit plasma. The influencing parameters on the extraction efficiency were optimized using experimental design methodology. The optimized conditions were found to be; supported liquid membrane: a mixture of 1‐octanol and 2‐ethyl hexanol (1:1) containing 10% v/v di(2‐ethylhexyl) phosphate, applied voltage: 5 V, extraction time: 5 min, pH of the donor phase: 4.5 and pH of the acceptor phase: 1.0. The optimized method was validated for linearity, intraday and interday precision, and accuracy in rabbit plasma. The range of quantification for both peptides was 0.5–1000 ng/mL with regression coefficients higher than 0.994. Relative recoveries of leuprolide and triptorelin were found to be 80.3 and 75.5%, respectively. Limits of quantification and detection for both peptides were found to be 0.5 and 0.15 ng/mL, respectively. The validated method was successfully applied to pharmacokinetic study of the 1‐month depot formulations of each peptide after subcutaneous administration to rabbits.     

A sensitive high performance liquid chromatography microassay for bemoradan, a novel cardiotonic agent, in plasma/serum using a semi-automatic liquid/solid extraction sample preparation system--AASP

A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP (Varian Associates, Sunnyvale, CA, USA), a semi-automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2-1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5-100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5-500 ng/mL.     

LC–MS/MS method for simultaneous determination of serum p‐cresyl sulfate and indoxyl sulfate in patients undergoing peritoneal dialysis

p‐Cresol sulfate (pCS) and indoxyl sulfate (IS) are protein‐bound uremic toxins that accumulate in patients with chronic kidney disease (CKD). They are closely associated with the mortality rate of CKD and morbidity of cardiovascular disease. In the present study, we established a rapid method for determination of pCS and IS by HPLC‐MS/MS in serum samples from 205 CKD patients undergoing peritoneal dialysis. In brief, serum was extracted by acetonitrile and spiked with hydrochlorothiazide. The prepared sample was eluted through HPLC column (Agilent Zorbax SB‐C₁₈, 3.5 μm, 2.1 × 100 mm) with a mobile phase of acetonitrile and 10 mm ammonium acetate solution (10:90, v/v) for subsequent detection of pCS and IS by MS/MS. The linearity ranged from 50 to 10,000 ng/mL for pCS (r > 0.99), and from 500 to 10,000 ng/mL for IS (r > 0.99). The lower limit of quantification was 50 ng/mL for pCS, and 500 ng/mL for IS. Relative standard deviation (RSD) of intra‐ and inter‐day precision was within ±15%. The results showed that pCS and IS levels were partially correlated with renal function in CKD patients, and IS was directly related to serum creatinine and estimated glomerular filtration rate.     

Determination of Atenolol in Pharmaceutical Preparations by Gas Chromatography with Flame Ionization and Mass Spectrometric Detection

The present work describes the methodology and validation of gas chromatography with flame ionization (FID) and mass spectrometric (MS) detection after derivatization with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for determination of atenolol with an internal standard (metoprolol) in pharmaceutical preparations. The linearity was established over the concentration range of 0.5–20 μg/mL for GC/FID and 12.5–500 ng/mL for GC/MS method. The intra- and inter-day relative standard deviation was less than 4.72 and 5.80%, respectively. Limit of quantification was determined as 500 ng/mL and 12.5 ng/mL for GC/FID and GC/MS, respectively. No interference was found from tablet excipients at the selected assay conditions. Developed GC/FID and GC/MS methods in this study are accurate, sensitive, and precise and can be easily applied to Tensinor tablet as pharmaceutical preparation.     

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.     

Determination of Ochratoxin A in wine at sub ng/mL levels by solid-phase microextraction coupled to liquid chromatography with fluorescence detection

Solid-phase microextraction (SPME), using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, interfaced with liquid chromatography-fluorescence detection (LC-FD) has been applied to the determination of Ochratoxin A (OTA) in wine samples. Compared to the most widely adopted extraction/clean-up procedure based on immunoaffinity columns (IAC), the solventless extraction is simpler and cost-effective, requiring the simple immersion of the fiber in diluted wine samples. Furthermore, a fast LC separation is achieved under isocratic conditions. The linear range investigated in wine was 0.25-8 ng/mL; at fortification levels of 0.5 and 2 ng/mL, within-day intra-laboratory precision (repeatability) values, expressed as RSD%, were 5.9 and 5.1, respectively, whereas between days (n = 4) precision was 8.5 and 7.1%, respectively. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 was 0.07 ng/mL; the limit of quantification (LOQ) calculated at S/N = 10 was 0.22 ng/mL, well below the European regulatory level of 2 ng/mL. The potential of the method has been demonstrated by the analysis of a number of different wine samples.     

Instrumental planar chromatographic method for determination of carbamazepine in human serum

An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid‐liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/μL showed correlation coefficient of 0.998. The intra‐assay and inter‐assay precision, expressed as the RSD, were in the range of 0.41–1.24% (n = 3) and 2.17–3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum.     

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography–MS/MS analysis: Application to a pharmacokinetic study

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI‐MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile–ammonium formate (10 mm , pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.     

Development of a liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of apicidin, a novel histone deacetylase inhibitor, in rat serum: application to a pharmacokinetic study

Apicidin, a fungal metabolite isolated from Fusarium pallidoroseum, is a cyclic tetrapeptide that exhibits potent anti-protozoal and anti-angiogenic activities. Although extensive studies have been recently conducted to examine the biological and pharmacological action, no information is available on the quantitative analysis of apicidin. To our knowledge, this study is the first to describe a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay method for the quantification of apicidin in rat serum. The method was validated to demonstrate the specificity, linearity, recovery, lower limit of quantification (LLOQ), accuracy, and precision. The multiple reaction monitoring was based on the transitions m/z 624.7 --> 84.3 and 372.1 --> 176.1 for apicidin and trazodone, respectively. The assay utilized a single liquid-liquid extraction and isocratic elution, and the LLOQ was 0.5 ng/mL using 0.1 mL of rat serum. The assay was linear over a range from 0.5-1000 ng/mL, with correlation coefficients >0.9994. The mean intra- and inter-day assay accuracy ranged from 99.9-101.5% and 94.8-102.1%, respectively, and the mean intra- and inter-day precision was between 2.7-5.9% and 1.6-11.5%, respectively. The developed assay method was applied to a pharmacokinetic study after intravenous injection of apicidin in rats at a dose of 1 mg/kg.