Sensitive determination of benzalkonium chloride in blood and tissues using high-performance liquid chromatography with solid-phase extraction

A sensitive and simple high-performance liquid chromatography assay for benzalkonium chloride (BZK) in biological samples was developed. The biological samples, spiked with domiphen used as an internal standard, were purified by solid-phase extraction. The major homologues of BZK in pharmaceutical products (C(12) and C(14)) were eluted at 24 and 36 min using a YMC-Pack CN column (4.6 x 250 mm, 5 microm) with a mobile phase, mixture of acetonitrile and sodium acetate buffer (48:52), and monitored at 254 nm. The most dominant component C(12) was selected as an indicator to quantify BZK, and adjustment was then done based on the proportion of C(12) in the BZK product. Good linearity was obtained in the range of 0.1-3 microg, and the limit of detection was 20 ng as a loaded amount on column. The recoveries of BZK in serum and tissues ranged from 54 to 90%. In a practical case, 0.16 microg/ml of BZK was quantified in serum collected several hours after accidental ingestion. The method is simple, sensitive and reliable for determining BZK levels in practical biological samples.     

Sensitive determination of specific radioactivity of positron emission tomography radiopharmaceuticals by radio high-performance liquid chromatography with fluorescence detection

A sensitive quality control method is often required in positron emission tomography (PET) radiopharmaceutical analysis due to the high specific radioactivity of synthetic products. The applicability of a radio high-performance liquid chromatography (HPLC) method with fluorescence detection was evaluated for a wide variety of PET radiopharmaceuticals. In 29 different radiopharmaceuticals studied, 20 compounds exhibited native fluorescence. These properties enabled sensitive determination of their chemical masses by direct fluorimetric detection after separation by HPLC. For some substances, detection limits were below nanograms per milliliter level, at least 40 times better than current UV absorbance detection. Sufficient reproducibility and linearity were obtained for the analysis of pharmaceutical fluid. Post-column fluorimetric derivatization was also established for the quantitative determination of FDG and ClDG in [(18)F]FDG samples. These methods could be applied successfully to the analysis of PET radiopharmaceuticals with ultra-high specific radioactivity.     

Sensitive determination of midazolam and identification of its two metabolites in human body fluids by column-switching capillary high-performance liquid chromatography/fast atom bombardment-mass spectrometry

Midazolam is a benzodiazepine and is widely prescribed for preanesthesia or general anesthesia. Overdose or intoxication cases of midazolam have been reported. In Japan, smuggled midazolam tablets could be involved in some criminal cases. Midazolam and its two metabolites were extracted by the solid-phase extraction method using Bond Elut SCX cartridges. The compounds were analyzed by on-line capillary high-performance liquid chromatography/fast atom bombardment-mass spectrometry. Midazolam and its two metabolites were well separated on the chromatogram, and each mass spectra gave [M+H](+) ion as a base peak. Deuterium-labeled midazolam was synthesized as an internal standard; it has enabled precise and reproducible quantitation of midazolam in blood samples. The calibration curve showed excellent linearity in the range of 2-200 ng/ml in spiked serum. The detection limit was 300 pg/ml (signal-to-noise ratio=3). The whole blood and urine samples from the victim of a homicide case were analyzed, and the midazolam concentration in the whole blood was estimated to be 163 ng/ml. The present method should be useful in clinical and forensic toxicology, because of its high sensitivity and specificity.     

Resolving mtDNA mixtures by denaturing high-performance liquid chromatography and linkage phase determination

Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture. The reliability of the approach is based, in part, on the strong correlation between a change in the relative quantities of different DNA amplicons in a given mixture versus a change in the relative electrophoretic peak heights at mixed base positions. Using standard operating procedures previously validated for use in forensic laboratories, this approach enables sequence-specific fractionation of natural (heteroplasmic) or situational (multi-contributor) DNA mixtures prior to direct sequencing. As a novel approach for the rapid and accurate analysis of DNA mixtures, DHPLC may aid criminal investigators by making it possible to obtain definitive mitochondrial DNA results from otherwise challenging samples.     

HPLC法测定左卡尼汀氯化钠注射液中左卡尼汀的含量

目的建立测定左卡尼汀氯化钠注射液含量的HPLC法。方法采用Diamonsil C18（200mm×4.6mm,5μm）色谱柱,以庚烷磺酸钠-磷酸盐缓冲液-乙腈（555mg：955ml：55ml）为流动相,检测波长为225nm,流速1ml/min。结果左卡尼汀在200~2000ng范围内线性关系良好（r=0.9998）,平均回收率为97.88%,RSD为1.07%。结论本方法快速、准确,可用于注射用左卡尼汀的含量测定。     

Species identification of blood and bloodstains by high-performance liquid chromatography

Summary A reverse-phase high-performance liquid chromatographic method for species identification of blood and bloodstains is described. The method employs a 300 Å pore SynChropak RP-4 column and ternary solvents (acetonitrile-trifluoroacetic acid-water) and can not only identify a species by its characteristic chromatogram, but also simultaneously demonstrates that it is of blood origin by the existence of the heme peak. Deformations in chromatographic profiles obtained with older bloodstains were observed, but the retention times of heme and the major peaks showed only minor changes. The species could be identified from bloodstains at least 3 months old and the present method has the advantage of simplicity, speed and sensitivity in the practice of forensic science.     

Quality control studies of 99mTc-labeled radiopharmaceuticals by high-performance liquid chromatography

Efficacy of HPLC in the quality control of commonly-used ^99mTc-labeled radiopharmaceuticals has been evaluated using different columns. μ-Bondagel and μ-Bondapak columns were found to be suitable for the separation of ^99mTcO₄⁻ from ^99mTc-labeled HSA and HIDA, respectively. Both of these columns offered marginal utility for DTPA, MDP and PPi.     

PR—HPLC法测定安神补心丸中五味子甲素的含量

安神补心丸由丹参?五味子?石菖蒲?合欢皮等药组成,经水煎浓缩制成?具有养心安神等功能,用于阴血不足引起的心悸失眠?头晕耳鸣?中国国家药典2000年版安神补心丸定性鉴别项下规定了五味子甲素鉴别方法,但未规定其有效成分含量测定的方法?我们已测定了某些含五味子的中成药中五味子甲素?五味子乙素含量[1～3],鉴于五味子具有收敛固涩?益气生中津?补肾宁心等作用?是安神补心丸中的主要君药,因此,有必要建立其有效成分含量测定方法?以利于规范制剂,提高质量?作为药用的五味子有北五味子(Schisandra chinesis Buill)和南五味子(中华五味子Schisa…     

高效液相色谱法测定人血浆样品中的椒苯酮胺

目的：建立椒苯酮胺(JBAT)在血浆样品中的高效液相色谱测定方法以满足临床前动物药代动力学研究的需要。方法：血浆样品用乙酸乙酯提取，氮气流吹干，用pH3、76的0．253mmol/L磷酸缓冲液80μl溶解后，取50μl进样分析。SymmetryShield^TMRP18分析柱，流动相为乙腈、pH2、8的5mmol/L磷酸缓冲液和甲醇以一定体积配比，流速1ml/min；紫外检测波长330nm。结果：在所用HPLC条件下，生物样品中内源性物质不干扰JBAT的测定。用内标法测定JBAT的血浆浓度在8—20000ng/ml范围内线性关系良好，最低定量限为8ng/ml，方法的准确度在-1．0％-5．9％之间，日间精密度为10．8％-13.2％，日内精密度为4．5％-9．6％。方法平均回收率为70．2％。结论：本方法线性范围广、专属性强、准确、稳定。     

LC-MS法检测大鼠血浆中酒石酸美托洛尔的方法学研究

目的：建立准确、灵敏、可靠的LC-MS法检测大鼠血浆中酒石酸美托洛尔的浓度。方法：血浆样品用乙酸乙酯液-液萃取,内标选用盐酸普萘洛尔,液相色谱柱为ThermoODS-C18（5.0μm,100mm×2.1mm）,流动相为含0.1%甲酸的乙腈：甲醇：水（20：20：60,体积比）,流速为0.2ml/min;仪器选用Agilent公司1100LC/MSDSL型液相色谱-质谱联用仪,采用电喷雾离子化电离源（ESI）,选择性离子监测（SIM）方式进行正离子检测,用于定量分析的离子反应分别为m/z267→268（美托洛尔）和m/z259→260（普萘洛尔）。结果：在本实验条件下,美托洛尔与内标盐酸普萘洛尔分离良好,保留时间分别为1.79和4.01min,0.1～50ng/ml范围内线性关系良好（r2=0.99411）,提取回收率均大于80%,日内日间精密度均小于15%（n=5）。结论：该方法快速、准确、灵敏度高,专属性好,可用于酒石酸美托洛尔在大鼠体内药物代谢动力学的研究。     

Identification of fetal hemoglobin and simultaneous estimation of bloodstain age by high-performance liquid chromatography

Summary A method using reverse-phase high-performance liquid chromatography (HPLC) for the identification of fetal hemoglobin (Hb F) and the simultaneous estimation of bloodstain age is described. Umbilical cord and neonatal bloodstains can be differentiated from adult stains by the presence of -globin chains which are characteristic of Hb F. With this method, cord and neonatal blood could be distinguished from adult blood in stains up to 32 weeks old. The age of the stain was estimated from the ratio of the peak area of the -globin chain to that of heme on the same chromatogram. The ratio decreased gradually with an increase in the age of the stain up to 20 weeks old. Studies performed at each time period revealed no significant difference in the ratios of cord and neonatal bloodstains or in the ratios of cord and adult stains. The regression equation calculated from the ratios (y) and the ages of stains in weeks (x) expressed logarithmically isy = 2.5758 – 0.2497 ln(x) and the coefficient of correlation is –0.7491 (n = 252,P < 0.001). The present method, having the advantages of simplicity, speed and sensitivity, should be of great value to forensic science.     

Analysis of paraquat,diquat and two diquat metabolites in biological materials by high-performance liquid chromatography

The determination of paraquat, diquat and two metabolites of diquat in biological materials was developed using high-performance liquid chromatography combined with UV and fluorescence detectors. Paraquat, diquat and internal standards (ethyl viologen and o-acetamidophenol) were detected by the UV detector. Diquat-monopyridone and diquat-dipyridone were monitored by the fluorescence detector. Paraquat, diquat, diquat-monopyridone and ethyl viologen were effectively extracted from blood, liver and brain, using a Sep-Pak C(18) cartridge. Diquat-dipyridone and o-acetamidophenol were extracted with methanol. Paraquat and diquat at a concentration range of 0.1-10 microg/ml (or g), and diquat-monopyridone and diquat-dipyridone at a concentration range of 0.01-1 microg/ml (or g) in biological material were determined with high accuracy and precision. The detection limits of paraquat, diquat, diquat-monopyridone and diquat-dipyridone were 1, 1, 0.02 and 0.02 ng, respectively, as an injection amount. This method was applied for toxicological examination of a case of suspected paraquat and diquat intoxication. The analysis of the metabolites of diquat was helpful for the estimation of the elapsed time from ingestion to death.     

HPLC法测定番石榴叶、苦瓜干和苦丁茶中槲皮素的含量

目的建立测定番石榴叶、苦瓜干和苦丁茶中槲皮素含量方法。方法采用DiamonsiLTM C18（250mm×4.6mm,5μm）,以甲醇-0.5%磷酸溶液（58：42）为流动相,检测波长为372nm,流速：1.0ml/min。结果槲皮素在1.064～10.64μg/ml之间成良好线性关系（r=0.9996）;番石榴叶、苦瓜干和苦丁茶的平均回收率分别为99.92%（RSD=0.4%）、102.6%（RSD%=0.7%）和102.9%（RSD%=0.1%）。结论该方法准确、灵敏,可作为槲皮素HPLC分析的一个有效的方法。     

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48 h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15 mg kg⁻¹. Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000 cps for 5 mg kg⁻¹, 6 h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7 min) detectable only after injection of at least 15 mg kg⁻¹. Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.     

Simultaneous determination of nicotine and cotinine in various human tissues using capillary gas chromatography/mass spectrometry

Summary A reliable and sensitive method for the simultaneous determination of nicotine and cotinine concentrations in various human tissues was developed using capillary gas chromatography/mass spectrometry. Nicotine and cotinine were extracted using a 3-step solvent extraction procedure and quinoline as an internal standard. Quantification was carried out by single ion monitoring using ions of m/z 133 for nicotine, m/z 176 for cotinine and m/z 129 for quinoline. The lower limit of detection was 5 ng/g for nicotine and 10 ng/g for cotinine, in each tissue sample. The calibration curves of various tissues were linear in the concentration range from 5–1,200 ng/g for nicotine and 10–1,500 ng/g for cotinine. The accuracy and precision of this method were examined using human tissues and the results were satisfactory. The distribution of nicotine and cotinine was measured in tissues from 10 human autopsies. Nicotine was detected in every tissue examined at a level seen in habitual smokers. The nicotine concentration was high in the liver, kidney, spleen and lung, and low in adipose tissue. The cotinine level was highest in the liver. The tissue/blood concentration ratios of nicotine and cotinine were most stable in skeletal muscle, where the level of these drugs was close to that in whole blood. Skeletal muscle is, therefore, considered to be the most suitable tissue sample for toxicological examination, when acquisition of blood samples is not feasible.     

Determination of some antiallergic drugs in human plasma by direct-injection high-performance liquid chromatography-tandem mass spectrometry

A detailed procedure for analysis of four antiallergic drugs, ketotifen, olopatadine, cetirizine, and ibudilast, in human plasma by high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS-MS) using a new polymer column (MSpak GF), which enables direct injection of crude biological samples, is presented. The protein and/or macromolecule matrix compounds were first eluted from the column, while the drugs were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base peaks due to [M + H]⁺ ions by HPLC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]⁺ ion by HPLC-MS-MS. Quantification was made by selected reaction monitoring. The recoveries of the four antiallergic drugs spiked into plasma were 51.7–95.5%. The regression equations for the four antiallergic drugs showed excellent linearity in the range of 1–100 ng/ml of plasma. The detection limit was 0.5 ng/ml for all compounds. The intraday and interday precisions for the drugs in plasma were not greater than 9.5%. The data obtained from actual determination of the antiallergic drugs in human plasma after their oral administration are also presented for validation of the method.     

Facile determination of natural cannabinoids in cannabis products using a conventional fully porous particle column and isocratic high-performance liquid chromatography with diode-array detector

Forensic Toxicology -     

Sensitive determination of mianserin and setiptiline in body fluids by gas chromatography with surface ionization detection (GC-SID)

Tetracyclic antidepressants, mianserin and setiptiline, in human body fluids have been found measurable with high sensitivity by gas chromatography (GC) with surface ionization detection (SID). The compounds in human whole blood or urine samples were extracted by solid-phase extraction using Bond Elut C(18) cartridges. The recoveries of both compounds from the body fluids were above 85%. For quantitation of mianserin, 25 ng of setiptiline was used as internal standard; and for quantitation of setiptiline, vice versa. The calibration curves for spiked whole blood or urine samples were linear in the range of 1.25-50 ng/ml. The detection limit of mianserin and setiptiline was about 1 ng/ml, which is comparable to those obtained by the previous GC-mass spectrometry methods. Our method seems very useful for determination of mianserin and setiptiline in forensic and clinical toxicology, because of high sensitivity, low background impurities and easy handling of the GC instrument.