Evaluation of a method for simultaneous quantification of codeine,dihydrocodeine, morphine,and 6-monoacetylmorphine in serum,blood, and postmortem blood

Summary A solid-phase extraction and gas chromatographic-mass spectrometric method for the simultaneous determination of codeine, dihydrocodeine, morphine, and 6-monoacetylmorphine in serum, blood or postmortem blood is described. The extraction technique allows the determination of free or total morphine (morphine plus morphine glucuronide). Experiments with spiked blood samples resulted in recoveries of 96.4% ± 4.2% for codeine, 95.8% ± 5.1% for dihydrocodeine, 90.3% ± 7.8% for 6-monoacetylmorphine and 92.5% ± 8.1% for morphine. Excellent linearity was obtained over the range 1–1500 ng/mL. The detection limit for all analytes is less than 1 ng/mL.     

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.     

Evaluation of a solid-phase extraction procedure for the simultaneous determination of morphine, 6-monoacetylmorphine,codeine and dihydrocodeine in plasma and whole blood by GC/MS

Different procedures of solid-phase extraction were re-examined and a new solid-phase extraction procedure was developed using gas chromatography-mass spectrometry for the simultaneous detection and quantitation of morphine, 6-monoacetylmorphine, codeine and dihydrocodeine in plasma and whole blood. The effects of different types of sorbent and buffer solutions on the recoveries and purity of the extracts were also studied. Some preparation techniques on whole blood samples were also investigated. The method developed using Chromabond C18 (100) with spiked plasma samples had good recoveries for all opiates of interest: morphine 93.1% ± 7.4%, 6-monoacetylmorphine 68.0% ± 6.7%, codeine 77.0% ±8.3% and dihydrocodeine 67.9% ± 8.4%. The detection limit of all compounds was less than 5 g/L. The blank plasma showed no interfering peaks in the GC/MS-analysis.     

Quantification of benzodiazepines in whole blood and serum

A high-performance liquid chromatography method for the determination of benzodiazepines and their metabolites in whole blood and serum using mass spectrometry (MS) and photodiode array (PDA) detection is presented. The combination of both detection types can complement each other and provides extensive case relevant data. The limits of quantification (LOQ) with the MS detection lie between 2 and 3 μg/l for the following benzodiazepines or metabolites: 7-amino-flunitrazepam, alprazolam, desalkyl-flurazepam, desmethyl-flunitrazepam, diazepam, flunitrazepam, flurazepam, α-hydroxy-midazolam, lorazepam, midazolam, nitrazepam, nordazepam and oxazepam, respectively 5 μg/l for lormetazepam and 6 μg/l for bromazepam. The LOQ of clobazam determined with the PDA detector is 10 μg/l. A convenient approach for determining the measurement uncertainty of the presented method—applicable also for other methods in an accreditation process—is presented. At low concentrations (<10 μg/l), measurement uncertainty was estimated to be about 50%, and at concentrations >180 μg/l, it was estimated to be about 15%. One hundred and twenty-eight case data acquired over 1 year are summarised.Presented 2003 at the workshop of the Society of Toxicological and Forensic Chemistry (GTFCh) in Zürich     

Bioanalytical method for simultaneous determination of benzodiazepines in vitreous humor using liquid chromatography-tandem mass spectrometry

The use of vitreous humor (VH) in forensic casework has been growing in the last years due to numerous advantages. Several compounds can be evaluated in this matrix, including benzodiazepines whose determination is essential due to their great availability and potential of dependance and misuse. Postmortem toxicological analyses are required to determine the influence of benzodiazepines in deaths. However, most of the analytical methods which determine these drugs in VH are laborious and time consuming. This article describes a simple method based on protein precipitation for the determination of eight benzodiazepines in VH samples. Samples were prepared through a protein precipitation method and analyzed by liquid chromatography tandem mass spectrometry. Solvent choice and sample and solvent volumes for precipitation were optimized using chemometric approaches. The method was validated for selectivity, lower limit of quantification (LLOQ), linearity, carryover, precision, bias, matrix effect and dilution integrity. In order to verify the applicability, 62 vitreous humor samples were analyzed. LLOQs were 1 ng/mL and calibration curves were linear from 1 to 25 ng/mL (r2 > 0,99) for all analytes. Bias, precision and dilution integrity results were satisfactory according to proper guidelines. Ionization suppression was significant with values ranging from 8 to 37%. Two samples from real cases were positive for diazepam with the following concentrations: 6.80 ng/mL and 47.68 ng/mL, approximately 10 times lower than those found in peripheral blood. The procedure described here can be used as a straightforward and low cost method for the quantitation of multiple benzodiazepines in VH.     

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.     

Antibody-mediated clean-up of blood for simultaneous HPLC determination of morphine and morphine glucuronides

For the interpretation of the concentration of morphine in blood samples of heroin consumers information about the concentration of the analgesic active morphine metabolite morphine-6-glucuronide is very important. Thus a simple but specific clean-up procedure based on immuno-affinity chromatography is presented for the extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from whole blood in cases of fatal heroin overdose. The preparation of the immunoabsorber by immobilization of antibodies against morphine-3-BSA and morphine-6-KLH with carbonyldiimidazole-activated trisacrylgel is described. The separation of the extracts is achieved by HPLC using native fluorescence detection. The limits of detection for this method are 10 ng for morphine and morphine glucuronides/g blood. The results for the concentration of morphine and morphine glucuronides in blood from seven cases of heroin overdose are presented. By calculating the quotients for the concentrations of morphine-6-glucuronide/morphine the time elapsed since the last intake of heroin is estimated. Received: 10 November 1996 / Received in revised form: 19 February 1997     

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material.In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol^® Reagent, Invitrogen; NucleoSpin^® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy^® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling.We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of ‘RNA integrity number’ (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates.By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin^® miRNA Kit excelling at DNA co-extraction and RNA results, respectively.Overall, there was no ‘best’ method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.     

A preliminary study on the stability of benzodiazepines in blood and plasma stored at 4° C

An approach to determine the stability of benzodiazepines and some of their metabolites (n = 13) by means of a routinely applied gas chromatographic method using electron capture detection was made in this preliminary study. Validation data of the method are given. Spiked blood and plasma samples were stored at 4° C and analysed at selected times up to 240 days. The concentrations of all analytes had decreased to at least 60% of the original levels at the end of the observation period. A clear pattern of breakdown could not be established. The data obtained suggest that results from long-term stored samples should be interpreted cautiously. Further investigations concerning the stability of drugs in blood and plasma samples, additional methods of identification and determination as well as the establishment of optimal storage conditions seem necessary. Received: 16 March 1997 / Received in revised form: 7 May 1997     

A simple,rapid and simultaneous analysis of complex volatile hydrocarbon mixtures in blood using gas chromatography/mass spectrometry with a wide-bore capillary column

Summary A screening method for detecting volatile hydrocarbons in blood has been developed using gas chromatography/mass spectrometry with a wide-bore capillary column and a headspace method. Toluene-d₈ and indan were used as the internal standards for quantitative analysis. Hydrocarbons with retention indices from 600 to 1200 were simultaneously and quantitatively detected in relatively low concentrations (0.01 g/m1) in reconstructed ion chromatography. This method could prove useful in forensic cases in which urgent examination of complex hydrocarbon mixtures, e.g. petroleum components, is required. Offprint requests to: K. Hara     

Analysis of amphetamine,methamphetamine, methylenedioxyamphetamine and methylenedioxymethamphetamine in whole blood using in-matrix ethyl chloroformate derivatization and automated headspace solid-phase microextraction followed by GC-MS

The in-matrix alkyl chloroformate derivatization method for amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), and methylene-dioxymethamphetamine (MDMA) was adapted for use with the whole blood matrix. This derivatization method was followed by automated headspace (HS)-solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) analysis. The sensitivity of this method, expressed as limit of detection, was approximately 10 ng/ml for these analytes tested in the blood matrix, which was sufficient to detect toxic concentrations of amphetamines in blood. The limit of quantitation for target analytes ranged from 0.05 to 0.2 μg/ml. The intraday precision and accuracy studies generally showed satisfactory results for all target compounds except MDA, in which a larger variation was observed. The in-matrix ethyl chloroformate derivatization of amphetamine, methamphetamine, MDA, and MDMA for HS-SPME was tested in other matrices such as stomach fluid, bile, thoracic cavity fluid, vitreous humor, brain, liver, spleen, and skeletal muscle. As a result, stomach fluid, thoracic cavity fluid, and vitreous humor showed SPME efficiencies higher than that of whole blood; however, this method was not suitable for solid tissue matrices under the present conditions.     

A simple and accurate method for measurement of the hemoglobin content in blood by colorimetric iron determination

Summary A simple and accurate determination of iron in blood involving acid digestion in a heating block is described. The digestion requires little attention and permits the handling of large numbers of samples. The diluted digest is used for the colorimetric determination of iron using o-phenanthroline, and the equivalent hemoglobin in blood is calculated. There was a good correlation between the results of the present method and the cyanmethemoglobin method with 21 standard blood samples containing 4.9–26.8 g/dl of hemoglobin.     

用高效液相色谱法同时测定血浆中苯妥英和卡马西平的浓度

建立了快速测定血浆或全血中苯妥英及卡马西平浓度的高效液相色谱（ＨＰＬＣ）法。样品经乙酸乙酯一步提取，用ＳｐｈｅｒｉｓｏｒｂＣ１８柱分析，流动相为甲醇一水一四氢呋喃（４５：５５：５，ｖ：ｖ：ｖ），二级管阵列检测器（ＤＡＤ）检测波长２１０±２ ｎｍ。本方法快速简便，有较好的精密度（ＲＳＤ苯妥英 １．９％～８．６％；卡马西平 ２．１％～ ７．８％）和较宽的线性范围（苯妥英 ０．４～ ４０．０ μｇ／ ｍｌ；卡马西平 ０．２～２０．０ μｇ／ ｍｌ），适合于临床血药浓度的监测和药代动力学的研究。     

Liquid chromatography tandem mass spectrometric method validation for the quantification of buprenorphine and norbuprenorphine in whole blood

A liquid chromatography tandem mass spectrometric (LC-MS/MS) method was developed for the determination of buprenorphine and norbuprenorphine present in whole blood using codeine-d3 as an internal standard. In this study, an alkaline buffer solution (pH 12) and a mixture of 9:1 ethyl acetate:cyclohexane were found efficient in extracting the target compounds. With the aid of a C18 column, a mobile phase containing 45:15:40 acidic acetonitrile: methanol: acidic water was also proved to be sufficient for resolving the target compounds in an isocratic manner. A positive electrospray ionisation mode (ESI+) was utilised for detection. The method presented imprecision < 12% for both analytes. Buprenorphine achieved an MDL = 1.2 ng/mL and an LOQ = 4.4 ng/mL, whereas norbuprenorphine obtained 0.9 ng/mL and 3.4 ng/mL for the respective measures. The regression lines showed a coefficient of determination, R² > 0.99 over the range between 10 and 400 ng/mL for both compounds. However, the preferred working range was decided to be 10 – 200 ng/mL for routine calibration. Within this routine range, the overall method achieved 83.5–103.1% accuracy at ng/mL level. This method is suitable for high throughput analysis as it is easy to use and affords rapid analysis.     

The effects of different intensity walking programs on serum blood lipids,high-sensitive C-reactive protein,and lipoprotein-associated phospholipase A2 in premenopausal women

Aim

This study examined the effects of 12 weeks of walking programs on serum lipids, high-sensitive C-reactive protein, and lipoprotein-associated phospholipase A2.

Methods

Twenty-six pre-menopausal women (30–49 years) completed 12 weeks of walking programs either at moderate or high intensity (50–55%, 70–75% maximum heart rate reserve, respectively). Estimated maximal oxygen consumption was assessed with a 2-km walking test; body composition, blood lipids, high-sensitive C-reactive protein, and lipoprotein-associated phospholipase A2 were measured before and after the study.

Results

Maximal oxygen consumption increased, favoring high-intensity group; body weights, percent body fat (p < 0.01) and body mass index (p < 0.05) decreased in both exercise groups. There were no significant changes in the measured blood lipids in any of the groups, except for a significant reduction in low-density lipoprotein-cholesterol in high-intensity group (p < 0.05). High-sensitive C-reactive protein and lipoprotein-associated phospholipase A2 levels reduced significantly in high-intensity (p < 0.01) and moderate-intensity (p < 0.05) groups, which were also different from the changes in the control group.

Conclusion

Walking programs with different intensity result in favorable changes; however, for protective effects against cardiovascular diseases, high-intensity walking may be advised due to greater reductions in low-density lipoprotein-cholesterol, and high-sensitive C-reactive protein and lipoprotein-associated phospholipase A2.     

军队不同年龄组男性干部血脂、血压、血糖检测结果比较

目的比较部队男性干部不同年龄组血脂、血压、血糖检测结果,找出差异,从而有针对性的做好健康教育。方法将2005年5月—2011年5月在我院疗养的3 026例男性干部根据年龄分为:老年前期组484例,老年组1 785例,高龄组757例,对各组血脂、血压、血糖异常分布及影响因素进行分析。结果老年前期组低高密度脂蛋白、高舒张压、饮酒、吸烟、缺乏运动的检出率分别为8.7%、23.8%、47.3%、28.1%、36.8%明显高于老年组的3.4%、16.2%、35.1%、18.1%、18.2%;而高收缩压、高空腹血糖、高餐后2h血糖及超重/肥胖的检出率明显降低,差异有统计学意义(P<0.01);高龄组高收缩压、缺乏运动的检出率分别为40.2%、27.3%明显高于老年组的32.6%、18.2%,高三酰甘油、高舒张压、高餐后2 h血糖,超重/肥胖,饮酒、吸烟的检查率明显降低,差异有统计学意义(P<0.01)。结论军队男性干部老年前期组低HDL-C、高DBP检出率高于老年组、高年组SBP、缺乏运动检出率明显增高;老年前组饮酒、吸烟、缺乏运动检出率增高。     

Continuous assessment of perfusion by tagging including volume and water extraction (CAPTIVE): A steady-state contrast agent technique for measuring blood flow,relative blood volume fraction,and the water extraction fraction

A new technique, CAPTIVE, that is a synthesis of arterial spin labeling (ASL) blood flow and steady-state susceptibility con trast relative blood volume imaging is described. Using a single injection of a novel, long half-life intravascular magnetopharma-ceutical with a high tissue:blood susceptibility difference (Δ χ) to ΔR1 ratio, changes in tissue transverse relaxivity (ΔR2 or ΔR2*) that arise from changes in blood volume were measured, while preserving the ability to measure blood flow using traditional T₁ -based ASL techniques. This modification permits the contin uous measurement of both blood flow and blood volume. Also, because the contrast agent can be used to remove the signal from intravascular spins, it is possible to measure the first-pass water extraction fraction. Contrast-to-noise is easily traded off with repetition rate, allowing the use of non-EPI scanners and more flexible imaging paradigms. The basic theory of these measurements, several experimental scenarios, and validating results are presented. Specifically, the PaCO₂-reactivity of mi-crovascular and total relative cerebral blood volume (rCBV), ce rebral blood flow (CBF), and the water extraction-flow product (EF) in rats with the new contrast agent MPEG-PL-DyDTPA is measured, and the values are concordant with those of previous literature. As an example of one possible application, continuous flow and volume measurements during transient focal ischemia are presented. It is believed that CAPTIVE imaging will yield a more complete picture of the hemodynamic state of an organ, and has further application for understanding the origins of the BOLD effect.     

Database of normal human cerebral blood flow, cerebral blood volume, cerebral oxygen extraction fraction and cerebral metabolic rate of oxygen measured by positron emission tomography with 15O-labelled carbon dioxide or water, carbon monoxide and oxygen: a multicentre study in Japan

Measurement of cerebral blood flow (CBF), cerebral blood volume (CBV), cerebral oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO₂) by positron emission tomography (PET) with oxygen-15 labelled carbon dioxide (C¹⁵O₂) or ¹⁵O-labelled water (H₂ ¹⁵O), ¹⁵O-labelled carbon monoxide (C¹⁵O) and ¹⁵O-labelled oxygen (¹⁵O₂) is useful for diagnosis and treatment planning in cases of cerebrovascular disease. The measured values theoretically depend on various factors, which may differ between PET centres. This study explored the applicability of a database of ¹⁵O-PET by examining between-centre and within-centre variation in values. Eleven PET centres participated in this multicentre study; seven used the steady-state inhalation method, one used build-up inhalation and three used bolus administration of C¹⁵O₂ (or H₂ ¹⁵O) and ¹⁵O₂. All used C¹⁵O for measurement of CBV. Subjects comprised 70 healthy volunteers (43 men and 27 women; mean age 51.8±15.1 years). Overall mean±SD values for cerebral cortical regions were: CBF=44.4±6.5 ml 100 ml^–1 min^–1; CBV=3.8±0.7 ml 100 ml^–1; OEF=0.44±0.06; CMRO₂=3.3±0.5 ml 100 ml^–1 min^–1. Significant between-centre variation was observed in CBV, OEF and CMRO₂ by one-way analysis of variance. However, the overall inter-individual variation in CBF, CBV, OEF and CMRO₂ was acceptably small. Building a database of normal cerebral haemodynamics obtained by the¹⁵O-PET methods may be practicable.     

A specific immunoassay for the determination of morphine and its glucuronides in human blood

The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with ¹²⁵I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection = 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described. Received: 22 December 1997 / Received in revised form: 23 March 1998