Mass Spectra and Cross-Contribution of Ion Intensity Between Drug Analytes and Their Isotopically Labelled Analogs - Benzodiazepines and Their Derivatives.

With GC-MS as the preferred method and isotopically labeled analogs (ILAs) of the analytes as the internal standards (ISs) of choice for quantitative determination of drugs/metabolites in biological specimens, one important aspect associated with chemical derivatization (CD) is that the CD products derived from the analyte and the selected IS must generate ions suitable for designating the analyte and the IS. These ions must not have significant cross-contribution (CC), i.e., ISs' contribution to the intensities of the ions designating the analytes, and vice versa. With this in mind, the authors have reviewed literature and information provided by manufacturers, searching for suitable CD reagents, CD methods, and ILAs of the analytes related to the following 18 benzodiazepines: oxazepam, diazepam, nordiazepam, nitrazepam, temazepam, clonazepam, 7-aminoclonazepam, prazepam, lorazepam, flunitrazepam, 7-aminoflunitrazepam, N-desalkylflurazepam, N-desmethylflunitrazepam, 2-hydroxyethylflurazepam, estazolam, alprazolam, α-hydroxyalprazolam, and α-hydroxytriazolam. These analytes and ILAs were derivatized with various derivatization groups, followed by GC-MS analysis. The resulting mass spectrometric data are systematically presented in two forms: (a) full-scan mass spectra; and (b) CC data of ion-pairs with potential for designating the analytes and their respective ILAs (candidates of ISs in quantitative analytical protocols). Many of these full-scan mass spectra are not yet available in the literature and should be of reference value to laboratories engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected in selected ion monitoring mode and systematically tabulated, making the data readily available for analysts searching for this important analytical parameter.

Mass Spectra and Cross-Contribution of Ion Intensity Between Drug Analytes and Their Isotopically Labelled Analogs - Common Opioids and Their Derivatives.

For the quantitation of most drugs and their metabolites, GC-MS is currently the preferred method and isotopically labeled analogs of the analytes are the internal standards (ISs) of choice. Under this analytical setting, chemical derivatization (CD) plays a critical role in the sample preparation process. In addition to meeting the conventional objectives of CD, products derived from the selected CD method must generate ions suitable for designating the analyte and the IS; these ions cannot have significant cross-contribution (CC), i.e., contribution to the intensity of the ions designating the analyte by the IS, and vice versa. With this in mind, the authors have reviewed literature and information provided by manufacturers, searching for suitable CD reagents, CD methods, and isotopically labeled analogs of the analytes related to the following 11 opioids: heroin, 6-acetylmorphine, morphine, hydromorphone, oxymorphone, 6-acetylcodeine, codeine, hydrocodone, dihydrocodeine, oxycodone, and noroxycodone. These analytes and ISs were derivatized with various derivatization groups, followed by GCMS analysis. The resulting MS data are systematically presented in two forms: (a) full-scan mass spectra; and (b) CC data of ion-pairs with potential for designating the analytes and their respective ISs. Many (if not most) of these full-scan mass spectra are not yet available in the literature and should be of reference value to laboratories engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected in selected ion monitoring mode and systematically tabulated, readily available for analysts searching for this important analytical parameter.

Determination of cocaine, its metabolites, pyrolysis products, and ethanol adducts in postmortem fluids and tissues using Zymark automated solid-phase extraction and gas chromatography-mass spectrometry.

Demonstrating the presence or absence of cocaine (COC) and COC-related molecules in postmortem fluids and/or tissues can have serious legal consequences and may help determine the cause of impairment and/or death. We have developed a simple method for the simultaneous determination of COC and the COC metabolites benzoylecgonine (BE), norbenzoylecgonine (NBE), ecgonine methyl ester (EME), ecgonine (E), and norcocaine (NCOC), as well as anhydroecgonine methyl ester (AEME) (a unique byproduct of COC smoking), cocaethylene (a molecule formed by the concurrent use of COC and ethanol) and their related metabolites, anhydroecgonine (AE), norcocaethylene (NCE), and ecgonine ethyl ester (EEE). This method incorporates a Zymark RapidTrace automated solid-phase extraction (SPE) system, gas chromatography/mass spectrometry (GC/MS) and 2,2,3,3,3-pentafluoro-1-propanol (PFP)/pentafluoropropionic anhydride (PFPA) derivatives. The lower limits of detection ranged from 0.78 to 12.5 ng/mL and the linear dynamic range for most analytes was 0.78-3200 ng/mL. The extraction efficiencies were from 26 to 84% with the exception of anhydroecgonine and ecgonine, which were from 1 to 4%. We applied this method to five aviation fatalities. This method has proven to be simple, robust and accurate for the simultaneous determination of COC and 11 COC metabolites in postmortem fluids and tissues.

Preparation of carboxyhemoglobin standards and calculation of spectrophotometric quantitation constants.

A method was developed for the preparation of carboxyhemoglobin (COHB) standards, which were stable for more than four months with the prepared control remaining within acceptable limits during this time. A mathematical equation was developed to more accurately determine the constants A and B used in the equation COHB% = 100[(C - B)/(A - B)], where B = 0% COHB peak ratio at 540 nm and 579 nm; A = 100% COHB peak ratio at 540 nm and 579 nm; and C = the peak ratio at 540 nm and 579 nm for the blood being analyzed. The following equations were developed to calculate A and B: B = Pavg - (P) [(Pavg - Navg)/(P - N)]; A = B + (Pavg - Navg)/(P - N), Pavg = average peak ratio 540/579 for the positive standard run on the spectrophotometer; P = average decimal concentration measured on the CO-OXIMETER for the positive standard; Navg = average peak ratio 540/579 for the negative standard; N = average decimal concentration measured on the CO-OXIMETER for the negative standard. The new equations provided results consistent with those obtained from a CO-OXIMETER.

DNA typing as a strategy for resolving issues relevant to forensic toxicology.

To investigate aircraft accidents, multiple postmortem biological samples of victims are submitted to the Civil Aeromedical Institute for toxicological evaluation. However, depending upon the nature of a particular accident, their body components are often scattered, disintegrated, commingled, contaminated, and/or putrefied. These factors impose difficulties with victim identification, tissue matching, and consequently authentic sample analysis and result interpretation. Nevertheless, these limitations can be overpowered by DNA typing. In this regard, three situations are hereby exemplified where DNA analysis was instrumental in resolving a tissue mismatching/commingling issue, pinpointing an accessioning/analytical error, and interpreting an unusual analytical result. Biological samples from these cases were examined for six independently inherited genetic loci using polymerase chain reaction (PCR) suitable for analyzing degraded DNA generally encountered in putrefied/contaminated samples. In the first situation, three of five specimen bags from one accident were labeled with two different names. DNA analysis revealed that one of these bags actually had commingled specimens, originating from two different individuals. Therefore, the sample was excluded from the final toxicological evaluation. In the second situation, an unacceptable blind control result was reported in a cyanide batch analysis. By comparing DNA profiles of the batch samples with those of the known positive and negative blind controls, it was concluded that the error had occurred during the analysis instead of accessioning. Accordingly, preventive measures were taken at the analytical level. The third situation was related to the presence of atropine at toxic concentrations in the blood (318 ng/mL) and lung (727 ng/g) with its absence in the liver, spleen, and brain. DNA analysis of the blood and liver samples exhibited their common identity, ensuring that the submitted samples had indeed originated from one individual. The selective presence of atropine was attributed to its possible administration into the thoracic cavity by the emergency medical personnel at the accident site for resuscitation, but circulatory failure prevented its further distribution. These examples clearly demonstrate the applicability of the PCR-based DNA typing to enhance the effectiveness of forensic toxicology operation.

Exposures to carbon monoxide, hydrogen cyanide and their mixtures: interrelationship between gas exposure concentration, time to incapacitation, carboxyhemoglobin and blood cyanide in rats.

Carbon monoxide (CO) and hydrogen cyanide (HCN) are generated during aircraft interior fires in sufficient amounts to incapacitate cabin occupants. For typical post-crash and in-flight fires, minimum protection periods of 5 and 35 min, respectively, have been suggested for breathing devices to protect the occupants from smoke. Relationships of blood carboxyhemoglobin (COHb) and cyanide (CN-) levels to incapacitation have not been well defined for these gases. Therefore, time to incapacitation (ti) and blood COHb and CN- at incapacitation were examined in rats exposed to CO (5706 ppm for 5-min ti; 1902 ppm for 35-min ti), HCN (184 ppm for 5-min ti; 64 ppm for 35-min ti) and their mixtures (equipotent concentrations of each gas that produced 5- and 35-min ti). Blood CO and HCN uptakes were evaluated at the two concentrations of each gas. With either gas, variation in ti was higher for the 35-min ti than the 5-min ti The COHb level reached a plateau prior to incapacitation at both CO concentrations, and COHb levels at the 5- and 35-min ti were different from each other. Blood CN- increased as a function of both HCN concentration and exposure time, but CN- at the 5-min ti was half of the 35-min ti CN- level. The HCN uptake at the high concentration was about three times that at the low concentration. In the high concentration CO-HCN mixture, ti was shortened from 5 to 2.6 min; COHb dropped from 81 to 55% and blood CN- from 2.3 to 1.1 microgram ml(-1). At the low-concentration CO-HCN mixture, where ti was reduced from 35 to 11.1 min, COHb decreased from 71 to 61% and blood CN- from 4.2 to 1.1 microgram ml(-1). Any alteration in the uptake of either gas by the presence of the other was minimal. Our findings suggest that specific levels of blood COHb and CN- cannot be correlated directly with the incapacitation onset and that postmortem blood COHb and CN- levels should be evaluated carefully in fire victims.