Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications.

This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4',6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Current trends in drug abuse associated fatalities - Jordan, 2000-2004.

This study is the first study that addresses drug abuse associated fatalities in Jordan. It is aimed to give a close picture to the demography, toxicological data, manner, cause of death and other associated findings in such cases. Postmortem forensic pathology reports for all autopsies examined in the National Institute of Forensic Medicine were reviewed over a 5-year period and drug abuse associated deaths were selected. The study revealed that 44 cases (0.76%) out of the 5789 total autopsies were attributed to drug abuse associated deaths. The age range was from 20 to 60 years (mean+/-S.D.=32.7+/-7.2). More than 80% of cases were Jordanian males. The reported abuse substances as single drug or in combination were alcohol in 56.8%, morphine 36.4%, heroin 15.9%, benzodiazepines in 11.4% and cocaine in one body packer case. Surprisingly, neither a case with amphetamine or amphetamine analogue, nor with marijuana or methadone was recorded. In 75% of cases the death was accidental and only one case was reported to be suicidal, while in 18.2% and 4.5% death was due to sudden death and road traffic accidents, respectively. Regarding the cause of death, it was related to drug overdose in 50% of cases and in 34.1%, 11.4% and 4.5% of cases it was attributed to drug related medical complications, non-drug related complications, and trauma, respectively. Alcohol was mainly associated with accidental death; morphine and heroin were associated with drug overdose and abused through intravenous route. Injection marks were reported in 56.8% of cases and in 52.3% death occurred at home. This study confirmed the variation in the incidence and type of abused substances in Jordan compared with different countries.

Agricultural and horticultural pesticides fatal poisoning; the Jordanian experience 1999-2002.

A prospective autopsy study addressing fatal poisoning with agricultural and horticultural pesticides was undertaken in Jordan over a 4 year period. A total number of 140 deaths occurred during 1999-2002. The mean fatality rate was 0.68 case per 100,000 population and the age range was 2-55 years; mean 28.3 years with male to female ratio 1.03. The largest number of cases occurred in those 20-29 years (n=69, 49.3%) followed by the age group 30-39 years (n=34, 24.3%) and 40-49 years (n=17, 12.1%). Less than 3.0% of the total fatal poisoning was noticed in both children younger than 9 years of age and those in the age group 50-59 years, with no fatal poisoning in adults at the age 60 years and above. At least 64.3% of all pesticide fatalities were due to suicide with male: female ratio (1.37:1). Accidental and homicide poisoning resulted in 24.3% and 7.9% of the total fatalities, respectively; however, only five cases 3.6% of fatal poisoning were due to unknown pesticides. The main pesticide used was carbamates with 110 cases 78.6% followed by organophosphorus 23 cases 16.4%. The study showed that the present legislation on pesticides availability in Jordan failed to reduce the number of fatal pesticides poisoning since the number of fatal pesticides poisoning was increased from 25.3 to 35 cases per year over a 20 years period. Enforcement of a new legislation addressing the availability of agricultural and horticultural pesticides for self-harm, especially carbamates and organophosphorus, is the most important strategy in the long term to prevent fatal pesticides poisoning in Jordan.

Development of a screening method for the most commonly abused anticholinergic drugs in Jordan; trihexyphenidyl, procyclidine and biperiden.

A sensitive and rapid method for the simultaneous determination of three commonly abused anticholinergic drugs in Jordan; trihexyphenidyl, procyclidine, and biperiden in plasma and urine has been developed using solid phase extraction and GC-MS. Linearity was established from therapeutic to fatal concentrations of the three drugs; 5-300 ng/ml in plasma, with correlation coefficient r(2) > or = 0.9978 and 10-800 ng/ml in urine r(2) > or = 0.9993. Recoveries were in the range of 86-92% and intra-day and inter-day relative standard deviations (n = 6) were in the range of 6.6-10.3% for the three drugs at three different concentrations in plasma and urine. The base peak m/z 98 for trihexyphenidyl, m/z 84 for procyclidine, and m/z 98 and 218 for biperiden, and m/z 339 for papaverine (internal standard) were monitored at selective ion monitoring; their retention times were 8.10, 8.67 and 8.92 min, respectively, and 14.79 min for the internal standard with analysis time of 16.75 min. The limit of detection of 0.5 ng/ml was attained for trihexyphenidyl and procyclidine, while for biperiden 2.0 and 1.0 ng/ml in spiked plasma and urine, respectively. This method has been applied to forensic and authentic samples taken from abuser and patients using these drugs. The method will offer the clinicians and the legal authority the right diagnosis regarding the anticholinergic involved in any case of abuse with less than 1 h per sample (plasma or urine) from the time of receiving.

Nicotine content in tobacco used in hubble-bubble smoking.

OBJECTIVE: To determine the nicotine content of commonly used tobacco in hubble-bubble (HB) and compare it with that found in cigarettes. METHODS: Analysis of nicotine content of 13 commercial brands of HB tobacco was carried out using gas chromatography over an 8 month period; November 2002 through to June 2003 at the Toxicology Laboratory, University of Jordan. RESULTS: A wide variation in nicotine content in all brands was noticed with an average of 8.32 mg/g tobacco, range (1.8-41.3 mg/g). The average nicotine content in each HB head (20 gm) of unflavored tobacco was (713 mg/head) and flavored tobacco has (67 mg/head). One head of unflavored tobacco has nicotine equivalent to 70 regular cigarettes. The addition of adulterations; honey, glycerin and other flavors in the process of preparing the flavored (Muasel) tobacco contributes to lowering the nicotine content in each gram of flavored tobacco. Smoking one head of flavored (Mua'sel) tobacco which contains on average one third of nicotine presented in 20 cigarettes (204 mg/pack) usually resulted in a higher plasma nicotine level by 20%. CONCLUSION: Hubble-bubble smokers are not at a lesser risk from smoking than cigarette smokers in relation to nicotine dependence. The inability of water to trap significant amounts of chemical substances present in tobacco (especially nicotine) and the danger which might result from the combustion of additives like; glycerin, honey and other flavors could be a very important factor to extrapolate the damage resulting from HB smoking.

Determination of tramadol in hair using solid phase extraction and GC-MS.

Tramadol is a centrally acting synthetic analgesic with mu-opioid receptor agonist activity, it is a widely prescribed analgesic used in the treatment of moderate to severe pain and as an alternative to opiates. Tramadol causes less respiratory depression than morphine at recommended doses. Its efficacy and low incidence of side effects lead to its unnecessary prescribing in patients with mild pain. Tramadol was classified as a "controlled drug" long after its approval for use in Jordan. Analysis of drugs of abuse in hair has been used in routine forensic toxicology as an alternative to blood in studying addiction history of drug abusers. A method for the determination of tramadol in hair using solid phase extraction and gas chromatography-mass spectrometry (GC-MS) is presented, the method offers excellent precision (3.5-9.8%, (M)=6.77%), accuracy (6.9-12%, M=9.4%) and limit of detection 0.5 ng/mg. The recovery was in the range of 87-94.3% with an average of 90.75%. The calibration curve was linear over the concentration range 0.5-5.0 ng/mg hair with correlation coefficient of 0.998. The developed method was tested on 11 hair samples taken from patients using tramadol as prescribed by their physician along with other different drugs in treating chronic illnesses. Tramadol was detected in all hair samples at a concentration of 0.176-16.3 ng/mg with mean concentration of 4.41 ng/mg. The developed method has the potential of being applied in forensic drug hair testing. In Jordan, hair drug testing started to draw the attention of legal authorities which stimulated forensic toxicologists in recent years to develop methods of analysis of drugs known or have the potential to be abused.

Investigating the presence of a common drug of abuse (benzhexol) in hair; the Jordanian experience.

The analysis of hair is now accepted as a recognised alternative method for the determination of drug misuse. It has several advantages over the biological fluids; blood and urine, including collection of information regarding long-term drug use and determination of compliance with treatment programmes. In Jordan, the abuse of Artane (benzhexol hydrochloride) has been recognised as the most commonly abused drug among Jordanian youths. Hair samples were collected from nine patients (Male 25-55 years, M=39.11, SD=10.53, CV=26.93%). Samples were analysed for the presence of benzhexol and the toxicological analysis revealed the presence of benzhexol in all samples and its concentration ranged from 0.104 to 7.81 ng/mg hair. Solid phase extraction and GC-MS on selective ion storage (SIS) were used for extraction and detection of the drug with papaverine as external standard. The mass detector was operated at selective ion storage (SIS) to monitor the m/z of 98 and 218 for benzhexol and m/z 339 and 324 for the papaverine. The retention times of benzhexol and papaverine were 6.77 and 12.48 min, respectively. The method was linear in the range of 0.5 to 40 ng/mg hair, with a mean coefficient of determination (R2=0.9982). The limit of detection was 0.04 ng/mg. The intra- and inter-day variations were 3.85% and 3.35%. Recovery was found to be above 90%.