Formation of an interfering substance, 3,4-dimethyl-5-phenyl-1,3-oxazolidine, during a pseudoephedrine urinalysis

During fatal aviation accident investigations, biosamples from the victims are submitted to the FAA Civil Aeromedical Institute (CAMI) for drug analysis. In the process of one such analysis by CAMI, an unknown substance was found in a urine sample. Simultaneous screening by thin layer chromatography (TLC) and gas chromatography/FID (GC/FID) suggested the presence of pseudoephedrine. A subsequent routine confirmation analysis of a separate urine aliquot by GC Fourier transform infrared (GC/FTIR) and GC mass spectrometry (GC/MS) indicated that the retention times of the unknown substance matched with those of pseudoephedrine. However, its infrared and mass spectra were different--the -OH and -NH groups were missing, a C-O-C group was present, and the molar mass was 12 atomic mass units (amu) more than that of pseudoephedrine. A subsequent literature search suggested that ephedrine-like amines react with aldehydes to form oxazolidines. Therefore, the 12-amu increase could be accounted for by condensation of pseudoephedrine with formaldehyde. Since this aldehyde is present in various grades of methanol and ethyl acetate, and these solvents were used during the solid-phase extraction, 3,4-dimethyl-5-phenyl-1,3-oxazolidine was synthesized by using (+)-pseudoephedrine HCl and formaldehyde. The analytical findings of the synthesized compound were consistent with those of the unknown interfering substance, confirming that it was the oxazolidine. Aldehyde contaminants in solvents or specimens can transform drugs of interest and may result in misidentification of a compound originally present in specimens. Therefore, chemicals used in analyses should be of the highest available purity, and a multi-analytical approach should be adopted to maintain a high degree of quality assurance.     

The Frye hearing in Florida: an attempt to exclude scientific evidence

State Supreme Courts require a minimum threshold of reliability and acceptance in the scientific community for all medical and similar evidence to be admitted at trial. In Florida and some other states, the courts adhere to what is known as the Frye standard, whereas in most states and in Federal Courts, it is the so-called Daubert standard. The jurisdiction of the present case is Hillsborough County (Tampa), Florida. Forensic pathologists seldom, if ever, are requested to participate in such hearings, unlike their toxicological and basic science colleagues who are more involved in research methodology and technical procedures. The burden is on the proponent of the evidence to prove the general acceptance of both the underlying scientific principle of the test and procedures used to apply that principle to the facts of the case at hand. The trial judge has the sole discretion to determine this question and general acceptance must be established by a preponderance of the evidence. The authors describe in detail a hearing in a case in which they were all involved. One author (WQS) had researched and documented the original scientific methodology in the literature. The situation involved a car and tractor trailer crash with the two occupants of the car dying of multiple trauma, whereas the truck driver was not injured. Autopsy of the auto driver revealed multiple injuries with exsanguination, and only vitreous humor and liver tissue, but not blood, were tested for ethyl alcohol. The estate of the driver of the automobile brought suit against the owner of the trucking company for wrongful death. The plaintiff requested a Frye hearing to question the reliability of testing other body specimens to translate to probable blood alcohol level. The testimony, submitted documents, and eventual decision by the judge are discussed.     

Population genetics of HPRTB, F13B, and LPL in Pernambuco, Northeast Brazil

One hundred thirty-four unrelated Northeast Brazilian individuals were typed for the HPRTB, F13B, and LPL short tandem repeats (STRs). DNA was amplified by specific primers and identified by silver staining of polyacrylamide gels. The allelic frequencies of these loci were in agreement with Hardy-Weinberg proportions. The most frequent alleles were HPRTB*13, F13B*10, LPL*10. The combined probability of paternity and the discrimination power of these 3 STRs were high, permitting their utilization for forensic science purposes.     

Paint and tape: collection and storage of microtraces of paint in adhesive tape

The collection and preservation of microtrace evidence with the aid of an adhesive tape is a method of choice in forensic science. This technique is rapid and easy and allows the concentration of microtraces on a carrier, which facilitates further investigations in the laboratory. Adhesive tapes are currently used to secure microtraces of fibers and glass, but hardly for traces of automotive paint and other lacquers for fear of interference with the analysis of binders. A collection of automotive paint consisting of original and repair lacquers collected by tape has been evaluated. After various times of storage within the tape, these samples were compared with untreated references by microscope FT-IR and microspectrophotometry (MSP). Another set of paints was collected in 1984, stored within the tape until 1995, and examined the same way. About 170 layers of lacquer with various types of binder were examined. With the exception of one clear lacquer no difference between treated samples and references was detected. This small difference observed could be correlated to the exposure to xylene (extractant) and was not caused by the storage within the adhesive tape.     

Loss of heterozygosity detected in a short tandem repeat (STR) locus commonly used for human DNA identification

Short tandem repeat (STR) markers are commonly used in basic genetic research and in human identification testing. Clinically, STRs can be used to study genetic alterations in tumors. A genetic deletion common to many types of cancer is referred to as the loss of heterozygosity (LOH). Numerous examples of LOH in cancer have been described and some have been mapped to areas located in close proximity to markers employed in human identity testing. Despite this fact, LOH has rarely been observed for STR loci commonly employed in forensic testing. Recently, for medico-legal purposes, we were asked to determine whether a tissue biopsy originated from a particular individual. For a reference source we assessed two specimens, one from normal tissue and one from cancerous tissue. When both reference specimens were used to generate DNA profiles, we observed LOH at one STR locus, D13S317. As demonstrated in other cancers only the cancerous biopsy demonstrated LOH. The forensic community should be cognizant of these unusual circumstances because, as identification of human DNA continues to be used more extensively, certain instances will arise in which reference material will not be readily available. In these situations, archived specimens may be employed as a reference source. Clinical specimens such as tissue biopsies should be used with caution if they have not been confirmed to contain normal tissue.     

Allelic frequencies for the HLA-DQA1, D1S80, HUMTHO1, HUMTPOX, HUMCSF1PO and HUMVWA loci in Cantabria (middle north Spain)

Allele frequencies for six DNA polymorphisms have been studied in a population sample from Cantabria (middle north Spain) using the polymerase chain reaction. The HLA-DQA1 locus was analyzed by the reverse dot-blot technique and the other five by polyacrylamide gel electrophoresis followed by silver staining. Six alleles were found for HLA-DQA1. 15 alleles for D1S80, 6 alleles for HUMTHO1 and HUMCSF1PO, 7 for HUMTPOX and 8 alleles for HUMVWA. The 21 repeat allele in HUMVWA had not previously been reported in a Spanish population. The genotype distributions met Hardy-Weinberg expectations for all the systems and some statistical parameters of forensic interest were calculated. Comparisons with other populations revealed significant differences for HLA-DQA1, HUMVWA and HUMTHO1, with interracial differences being more pronounced than between Spanish populations. The HUMVWA system showed the highest forensic efficiency of the six polymorphisms studied.     

Population data on the X chromosome short tandem repeat locus HumHPRTB in two regions of Germany

This report contains the results of two population studies on the X chromosome STR HumHPRTB carried out in a Northern and a Southern region of Germany. The numbers of unrelated individuals were 443 and 335, respectively. Eight alleles (alleles 9 to 16) were found. In female individuals 29 different genotypes were encountered. In German populations the HumHPRTB STR was characterized by the following data: PIC = 0.750; HET = 0.769: MEC = 0.556. Allele distribution met the Hardy-Weinberg expectations. The Northern and Southern populations did not show any significant differences.     

Potency trends of delta9-THC and other cannabinoids in confiscated marijuana from 1980-1997

The analysis of 35,312 cannabis preparations confiscated in the USA over a period of 18 years for delta-9-tetrahydrocannabinol (delta9-THC) and other major cannabinoids is reported. Samples were identified as cannabis, hashish, or hash oil. Cannabis samples were further subdivided into marijuana (loose material, kilobricks and buds), sinsemilla, Thai sticks and ditchweed. The data showed that more than 82% of all confiscated samples were in the marijuana category for every year except 1980 (61%) and 1981 (75%). The potency (concentration of delta9-THC) of marijuana samples rose from less than 1.5% in 1980 to approximately 3.3% in 1983 and 1984, then fluctuated around 3% till 1992. Since 1992, the potency of confiscated marijuana samples has continuously risen, going from 3.1% in 1992 to 4.2% in 1997. The average concentration of delta9-THC in all cannabis samples showed a gradual rise from 3% in 1991 to 4.47% in 1997. Hashish and hash oil, on the other hand, showed no specific potency trends. Other major cannabinoids [cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC)] showed no significant change in their concentration over the years.