The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.     

Isolation, extractive concentration, and determination of caffeine in the studies of blood plasma

The optimal conditions for the isolation of caffeine from human blood by means of acetone extraction are described with special reference to the peculiarities of extraction from aqueous solutions. The possibility of concentration and purification of caffeine from blood plasma using acetone and aceton-chlorophorm mixture (2:8) as the solvents is illustrated. In addition, purification by silica-gel thin layer chromatography is discussed. Thin layer chromatography, UV-spectrophotometry, and high performance liquid chromatography are considered as potential methods for the identification and quantitative determination of caffeine.     

Rapid detection of caffeine in blood by freeze-out extraction

A new method for the detection of caffeine in blood has been proposed based on the combination of extraction and freezing-out to eliminate the influence of sample matrix. Metrological characteristics of the method are presented. Selectivity of detection is achieved by optimal conditions of analysis by high performance liquid chromatography. The method is technically simple and cost-efficient, it ensures rapid performance of the studies.     

血中安定及其代谢物的酶水解研究

目的考察木瓜蛋白酶水解血中安定及其代谢物蛋白结合物的酶解条件,提高安定的提取率。方法采用正交试验确定酶解的最优条件,检材经蛋白酶水解,固相萃取后,应用LC-MS／MS方法进行检测,运用保留时间和MRM（多离子反应监测）方式来对血中安定及其代谢物进行定性定量分析。结果安定、去甲安定、去甲异安定、羟基安定和去甲羟基安定的最佳酶解条件分别为55℃,2．5h,pH7．0,8000U;50℃,1h,pH7．5,8000U;50℃,1h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U;50℃,1．5h,pH7．5,8000U。结论酶水解后的血液中安定及其代谢物检出量明显增加。     

Analytical approach to determine ticlopidine in post-mortem blood

A new and sensitive method to determine ticlopidine in whole human blood using proadifen as the internal standard (IS) is described. The analyte and IS were extracted by solid-phase extraction using Oasis HLB cartridges, and the extracts were analyzed by gas chromatography-electron impact ionisation-mass spectrometry (GC/EI-MS). Calibration curves were established daily in spiked blood samples using a non-linear calibration model, between 0.01 and 4.5 microg/mL. The correlation coefficients were higher than 0.995. Precision and accuracy fulfilled the internationally accepted criteria (coefficients of variation were less than 9%, and the measured concentrations were within +/-7% of the true value). Limits of detection and quantitation were respectively, 3 and 10 ng/mL. No interfering substances were detected by analysis of 10 blank blood samples of different origin. Mean recovery, calculated at three concentration levels, was 71%. Because of its simplicity and speed, the proposed method can be applied in the determination of this inhibitor of platelet aggregation in whole blood samples, and is suitable for application in toxicology routine analysis.     

Optimal conditions for extraction of "caffetin" and "saridon" tablet components from aqueous solutions

Using a mathematical method of experiment planning (Latin square), the authors suggest the optimal conditions for extraction of propifenasone and paracetamol, the basic components of caffeine and saridon tablets, from water solutions: extraction with ethylacetate (pH 2) for 5 min in the presence of an electrolyte (sodium chloride or ammonium sulfate) quantum satis. The possibility of extraction of caffeine and codeine under these conditions was tested. When extracting the components of caffeine and saridon tablets, paracetamol, propifenasone, and caffeine should be extracted with ethylacetate at pH 2 and codeine by chloroform at pH 10.     

GC法检测血液和尿液中甲基苯丙胺和咖啡因

目的建立同时测定血、尿中甲基苯丙胺和咖啡因含量的方法。方法应用GC／NPD技术,以4-苯基丁胺为内标,直接碱化,用氯仿提取,三氟乙酸酐衍生化,8CB熔融石英毛细管柱（30m×0．25mm×0．25μm）分析。结果生物样品中甲基苯丙胺与咖啡因在0．012—7．5μg/mL浓度范围内线性关系良好,检测限（S／N=3）依次为1．2ng／mL,0．6ng／mL（血）;1．6ng／mL,0．8ng／mL（尿）。苯丙胺在0．017—10．0μg／mL浓度范围内线性关系良好,检测限为1．6mg／mL（血）,3．2ng／mL（尿）。所有样本回收率均大于85％。结论本方法准确、灵敏,适用于血、尿中甲基苯丙胺及其代谢物苯丙胺的三氟乙酸酐衍生化物和咖啡因的同时检测,为判定滥用毒品种类、追查毒品来源以及研究生物体内甲基苯丙胺和咖啡因的交互影响提供了检测手段。     

Failure to detect elevated levels of carboxyhemoglobin in infants dying from SIDS

Carboxyhemoglobin (COHb) levels were determined in stored blood samples from 91 infants diagnosed to have died from the sudden infant death syndrome (SIDS) (0.59+/-0.41%, excluding one outlying value of 10.83%); 48 age-matched controls (0.53+/-0.38%); and three individuals who died from fire related causes (41+/-20%). No statistical differences in COHb levels were detected between blood from SIDS and control infants (p = 0.43).     

Disposition of citalopram in biological specimens from postmortem cases

Citalopram is a bicyclic phthalate compound approved in 1998 by the U.S. Food and Drug Administration for the treatment of depression. It is a highly selective serotonin reuptake inhibitor that appears to have little effect on noradrenaline or dopamine reuptake. Since this drug has only recently been released on the U.S. market, information regarding therapeutic, toxic, and lethal concentrations is sparse. This study reports the detection of citalopram in 22 postmortem cases. Citalopram was identified and quantitated by capillary column gas chromatography with nitrogen phosphorus detection after basic liquid-liquid extraction. Confirmation was achieved by full scan electron impact gas chromatography/mass spectrometry. In the 22 cases studied, heart blood citalopram concentrations ranged from 0.09 to 1.64 mg/L (n = 22, mean +/- SD = 0.51+/-0.43, median = 0.34); femoral blood concentrations ranged from 0.09 to 0.76 mg/L (n = 14, mean +/- SD = 0.34+/-0.23, median = 0.28); and urine concentrations ranged from 0.05 to 276.00 mg/L (n = 13). Liver was analyzed in three cases with citalopram concentrations ranging from 2.22 to 8.08 mg/kg. The average heart blood/femoral blood ratio was 1.26 (range 0.75 to 1.98, n = 14). In each case, the cause of death was not considered to be related to citalopram toxicity. These data may therefore provide a basis for establishing post mortem citalopram concentrations following therapeutic doses.     

A multi-chemical death involving caffeine, nicotine and malathion

Death of a 21-year-old man who was found in a shower stall in his residence is described in the study. At the scene, a 3/4 filled blue glass bottle labeled "Black Leaf 40" (an insecticide containing nicotine), a white plastic pitcher 1/3 full of thick white fluid, a beer mug 1/4 full of thick white fluid, and an empty carton of milk were found. In addition, a can of malathion and an empty bottle labeled caffeine also were found in the vicinity. Autopsy was performed, and the gross examinations of organs revealed no specific findings to account for the death. However, marked congestion in lung, liver, spleen and kidney were noted at microscopic level. Autolytic degenerative changes were also observed in stomach, small bowel and colon. Toxicological analyses of the autopsy samples (blood, urine, liver and gastric contents) revealed the presence of caffeine and nicotine in each sample. Malathion was found to be present only in gastric content. Caffeine and nicotine were analyzed by utilizing gas liquid chromatography-nitrogen phosphorus detector, while malathion was by gas liquid chromatography-flame photometric detector. Analyses of the fluids from the bottle, pitcher and mug disclosed the presence of nicotine in the concentrations of 17.8%, 3.7% and 5.7% (w/w), respectively. The fluids from the pitcher and mug also contained 2.7-2.9% malathion. Results conclude the death was associated with caffeine, nicotine and malathion.     

Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

Kinetics of ethanol in biological sections

Ethanol concentration in alveolocapillary blood (ACB), venous blood (VB), capillary blood (CB), saliva and urine was measured in healthy men and women aged 19-45 years 20, 40, 60, 90, 120, 180, 240 and 300 min after a single intake of 20% ethanol solution in soda water in a dose 0.8 g/kg body mass. Two types of kinetic curves were established. Calculations with Vidmark equation for different biomedia were made. Ethanol levels in all BM studied coincided in the resorption phase. In the elimination phase, ethanol concentration forms a sequence: ACB < saliva < VB < urine. Correlations and correlation coefficients of ethanol concentrations in different BM were estimated. The ethanol concentration correlation urine/ACB 1.71 +/- 0.15 and VB/ACB 1.45 +/- 0.07 is proposed for use in tests for alcohol intoxication.     

Forensic and chemical determination of methane in cadaveric samples

Conditions were elaborated for the determination of methane, through gas chromatography, in blood and the lung by using a 2% methanol solution as an internal standard and with the below gas-chromatography column being applied: dimensions--200 x 0.3 cm, 15% di-2-ethylhexyl sebacate on Dinochrome II (0.16 x 0.21 mm) at 110 0 degree C. The detector is of the flame-ionization type. The normal content of methane was determined for blood and for the lung--0.122 +/- 0.0074 microgram/ml and 0.20 +/- 0.04 microgram/g, respectively. Biological samples from cadavers of other persons who died due to trauma were suggested for use as controls. The method was used to examine expertise objects, blood and the lung from the cadavers of peoples who died in fire.     

A large-scale study of the relationship between blood and breath alcohol concentrations in New Zealand drinking drivers

Blood alcohol concentrations (BAC) and corresponding breath alcohol concentrations (BrAC) were determined for 21,582 drivers apprehended by New Zealand police. BAC was measured using headspace gas chromatography, and BrAC was determined with Intoxilyzer 5000 or Seres Ethylometre infrared analysers. The delay (DEL) between breath testing and blood sampling ranged from 0.03 to 5.4 h. BAC/BrAC ratios were calculated before and after BAC values were corrected for DEL using 19 mg/dL/h as an estimate of the blood alcohol clearance rate. Calculations were performed for single and duplicate breath samples obtained using the Intoxilyzer (groups I-1 and I-2) and Seres devices (groups S-1 and S-2). Before correction for DEL, BAC/BrAC ratios for groups I-1, I-2, S-1, and S-2 were (mean+/-SD) 2320+/-260, 2180+/-242, 2330+/-276, and 2250+/-259, respectively. After BAC values were adjusted for DEL, BAC/BrAC ratios for these groups were (mean+/-SD) 2510+/-256, 2370+/-240, 2520+/-280, and 2440+/-260, respectively. Our results indicate that in New Zealand the mean BAC/BrAC ratio is 19-26% higher than the ratio of the respective legal limits (2000).     

Nicotine and cotinine levels in body fluids of smokers who committed suicide

Cigarette smoking is associated with a higher risk for suicide. The present study was conducted on the hypothesis that suicide smokers show higher nicotine and cotinine levels in blood and urine than non-suicide smokers. We determined nicotine and cotinine levels in blood and urine of 87 deceased individuals (18 suicides and 69 non-suicides) by gas chromatography. The smoking rate was 77.8% for individuals who committed suicide and 42.0% for those who did not commit suicide. Average nicotine and cotinine levels in blood were significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 93.2+/-46.6 ng/ml versus 25.8+/-14.4 ng/ml, p<0.0001 and cotinine: 378+/-235 ng/ml versus 201+/-137 ng/ml, p<0.005). Average levels of urinary nicotine and cotinine were also significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 1980+/-2210 ng/ml versus 394+/-376 ng/ml, p<0.005 and cotinine: 1170+/-1330 ng/ml versus 414+/-290 ng/ml, p<0.05). Twenty-six decedents were intoxicated with alcohol, and they included 7 suicides (7 smokers) and 19 non-suicides (15 smokers). Our data suggest that cigarette smokers who commit suicide smoke more heavily than other cigarette smokers.     

Psychopathology and weapon choice: a study of 103 perpetrators of homicide or attempted homicide

Bone marrow (BM) analysis is of forensic interest in postmortem toxicological investigation in case of limited, unavailable or unusable blood samples. However, it remains difficult to determine whether a drug BM concentration is therapeutic or represents overdose, due to the lack of studies on this alternative matrix. Given the variations in BM composition in the body, sample location was suggested to be a relevant factor in assessing BM concentration. The aim of the present study was to compare postmortem caffeine concentrations in various BM sample locations and secondly to consider the correlation between BM and blood concentrations. Six BM samples (right and left side: proximal and medial femur and 5th rib) and a blood sample were collected from 21 forensic autopsies. Gas chromatography coupled to tandem mass spectrometry was performed. Blood caffeine concentrations ranged from 60 to 7591ng/mL. Femoral and rib BM concentrations ranged from 51 to 6171ng/g and 66 to 7280ng/g, respectively. Blood concentrations were always higher than BM concentrations. As a good correlation was demonstrated between blood and rib BM and between blood and the average of the four femoral BM concentrations, blood caffeine concentrations could be correctly extrapolated from BM concentrations. BM caffeine concentration was found to depend on sample location. Rib BM caffeine concentrations appeared to be systematically greater than averaged femur values and concentrations were much more variable between the 4 femur BM samples than between the 2 ribs. From a practical point of view, for caffeine analysis, rib BM appeared more relevant than femoral BM, which requires multisampling to overcome the concentration variability problem.     

Comparison of ethanol concentrations in venous blood and end-expired breath during a controlled drinking study

Concentration-time profiles of ethanol were determined for venous whole blood and end-expired breath during a controlled drinking experiment in which healthy men (n=9) and women (n=9) drank 0.40-0.65 g ethanol per kg body weight in 20-30 min. Specimens of blood and breath were obtained for analysis of ethanol starting at 50-60 min post-dosing and then every 30-60 min for 3-6 h. This protocol furnished 130 blood-breath pairs for statistical evaluation. Blood-ethanol concentration (BAC, mg/g) was determined by headspace gas chromatography and breath-ethanol concentration (BrAC, mg/2l) was determined with a quantitative infrared analyzer (Intoxilyzer 5000S), which is the instrument currently used in Sweden for legal purposes. In 18 instances the Intoxilyzer 5000S gave readings of 0.00 mg/2l whereas the actual BAC was 0.08 mg/g on average (range 0.04-0.15 mg/g). The remaining 112 blood- and breath-alcohol measurements were highly correlated (r=0.97) and the regression relationship was BAC=0.10+0.91BrAC and the residual standard deviation (S.D.) was 0.042 mg/g (8.4%). The slope (0.91+/-0.0217) differed significantly from unity being 9% low and the intercept (0.10+/-0.0101) deviated from zero (t=10.2, P<0.001), indicating the presence of both proportional and constant bias, respectively. The mean bias (BAC - BrAC) was 0.068 mg/g and the 95% limits of agreement were -0.021 and 0.156 mg/g. The average BAC/BrAC ratio was 2448+/-540 (+/-S.D.) with a median of 2351 and 2.5th and 97.5th percentiles of 1836 and 4082. We found no significant gender-related differences in BAC/BrAC ratios, being 2553+/-576 for men and 2417+/-494 for women (t=1.34, P>0.05). The mean rate of ethanol disappearance from blood was 0.157+/-0.021 mg/(g per hour), which was very close to the elimination rate from breath of 0.161+/-0.021 mg/(2l per hour) (P>0.05). Breath-test results obtained with Intoxilyzer 5000S (mg/2l) were generally less than the coexisting concentrations of ethanol in venous blood (mg/g), which gives an advantage to the suspect who provides breath compared with blood in cases close to a threshold alcohol limit.     

Experimental studies on death by fire in automobiles and exhaust gas poisoning

Studies were made on the acid-base balance, blood gases, and carbon monoxide (CO), cyanide, and sulfur dioxide concentrations in the blood of albino rabbits that died from automobile exhaust gas poisoning (group I) or fires in cars (complete combustion, group II; incomplete combustion, group III). In group I, the temperature and CO concentration increased gradually to 35 degrees C and 5.2% in 70 min. The animals died after 9 min, when the values were 20 degrees C and 5.2%, respectively. In group II the animals died after 9 min, when the values were 55 degrees C and 1.95%, respectively. In group III, the temperature was very high (870 degrees C), but the CO concentration was not (0.6-1.3%) after 4 min. The animals died after 5 min. In all experimental groups, marked acidosis and hypoxemia were seen, but the CO2 tension (PCO2) was high, in contrast to previous studies on pure CO poisoning. In group I, the level of carboxyhemoglobin (CO-Hb) was significantly higher (91.2 +/- 3.4% in arterial blood, 87.5 +/- 8.1% in venous blood; p less than 0.01) than in groups II and III. Although the O2 tensions of venous and arterial blood (PvO2, PaO2) were very low, that of arterial blood was higher, suggesting that O2 was still being utilized in the tissues at the time of death. In group II, CO-Hb was high (57.7 +/- 16.0% in arterial blood, 61.2 +/- 20.6% in venous blood) and the acid-base balance indicated marked acidosis. In group III, the CO-Hb, PCO2 and cyanide levels in the blood were very high.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.