Amy P. Hart

Comparison of capillary electrophoresis and polyacrylamide gel electrophoresis for the evaluation of T and B cell clonality by polymerase chain reaction.

Polymerase chain reaction (PCR) technique is widely used in the diagnosis of lymphoma, and PCR amplification products are typically detected by polyacrylamide gel electrophoresis (PAGE). However, the identification of small clonal populations, or the distinction of clonal PCR products in a polyclonal milieu remains difficult, requiring technically demanding alterations to gel analysis. This study describes an alternative approach using a capillary electrophoresis (CE) system to produce an accurately sized electropherogram. A variety of patient samples were examined, including solid tissue, peripheral blood, bone marrow aspirates, and paraffin-embedded tissue. A total of 28 samples were evaluated by PCR for B-cell clonality by detection of immunoglobulin heavy chain gene rearrangement and 29 samples for T-cell clonality by detection of T-cell gamma locus gene rearrangement. Standard 10% PAGE analysis of PCR products was compared with CE. There was a 100% concordance in the assessment of both B-cell and T-cell clonality. Dilution studies with the SUP-B15 cell line showed a detection limit of 0.03% for B-cell clonality and 0.05% for T-cell clonality using CE, versus 0.2% to 1%, respectively for PAGE. Automated, fluorescent analysis of PCR products by CE seems to be at least equally as effective as gel-based analysis for the detection of clonal B-cell and T-cell populations. Moreover, CE offers superior resolution and improved sensitivity, thus representing a significant improvement over traditional gel electrophoretic techniques in these regards.

Lamotrigine analysis in plasma by gas chromatography–mass spectrometry after conversion to a tert.-butyldimethylsilyl derivative

Lamotrigine (lamictal) is a new anticonvulsant drug recently approved by the FDA for clinical use. Therapeutic monitoring of lamotrigine is useful for patient management (therapeutic range 1-4 microg/ml). Here we describe a gas chromatography-mass spectrometric identification and quantitation of lamotrigine after extraction from human serum and derivatization. Lamotrigine was extracted from alkaline serum with chloroform and derivatized with N-methyl-N-(tert.-butyldimethysilyl) trifluoroacetamide containing 2% tert.-butyldimethylchlorosilane. Oxazepam-d5 was used as an internal standard. The tert.-butyldimethylsilyl derivative of lamotrigine showed distinct molecular ions at m/z 483 and 485 as well as other peaks at m/z 426, 370 and 334 for unambiguous identification. The base peak was observed at m/z 199. Similarly, the tert.-butyldimethysilyl derivative of oxazepam-d5 showed molecular ions at m/z 519 and 521 along with other characteristic peaks at m/z 462, 376 and 318. For the analysis of lamotrigine, the mass spectrometer was operated in the selective ion monitoring mode. The within-run and between-run precisions were 4.3% (mean=3.01, S.D.=0.13 microg/ml) and 5.1% (mean=2.93, S.D.=0.15 microg/ml), respectively at a serum lamotrigine concentration of 3.0 microg/ml. The within-run and between-run precisions were 8.2% (mean=0.49, S.D.=0.04 microg/ml) and 10.6% (mean=0.47, S.D.=0.05 microg/ml), respectively at a serum lamotrigine concentration of 0.5 microg/ml. The assay was linear for serum lamotrigine concentrations of 0.5-20 microg/ml. The detection limit was 0.25 microg/ml. The assay was free from interferences from common tricyclic antidepressants, benzodiazepines, other common anticonvulsants, salicylate and acetaminophen.

A rapid cost-effective high-performance liquid chromatographic (HPLC) assay of serum lamotrigine after liquid-liquid extraction and using HPLC conditions routinely used for analysis of barbiturates

Lamotrigine (lamictal) is a new anticonvulsant drug approved by the FDA for clinical use. Therapeutic monitoring of lamotrigine is useful for patient management and avoidance of toxicity. The suggested therapeutic range is 1 to 4 micrograms/ml. The authors describe a simple high-performance liquid chromatographic (HPLC) method for analysis of lamotrigine from serum. Serum (0.5 ml) was alkalinized with borate buffer (pH 9.8). Lamotrigine and the internal standard thiopental were extracted with 10 ml of chloroform. After evaporation of the extract, the residue was reconstituted in the mobile phase (prepared by mixing 750 ml of potassium dihydrogen phosphate, 550 ml of deionized water, 430 ml of methanol, and 100 microliters of triethylamine as an ion pairing reagent) and injected into an LC-18 column (15 cm x 4.6 mm). The authors use this HPLC system routinely in their laboratory for the analysis of barbiturates. They demonstrated that the same system can be used for the analysis of lamotrigine. The within-run and between-run precisions of the lamotrigine assay were 1.63% (mean = 3.05, SD = 0.05 microgram/ml, n = 6) and 3.7% (mean = 2.97 micrograms/ml, SD = 0.11, n = 8). The assay was linear for serum lamotrigine concentrations of 0.5 microgram/ml to 20 micrograms/ml with a detection limit of 0.5 microgram/ml. The authors observed excellent correlation between serum lamotrigine concentrations measured by their assay and a reference laboratory in six patients receiving lamotrigine. Their assay is free from interferences from common tricyclic antidepressants, benzodiazepines, other common anticonvulsants, salicylate, and acetaminophen.

Autopsy artifact created by the Revivant autopulse resuscitation device

In certain cases, the evaluation and correct identification of resuscitative artifacts is critical to the correct diagnosis and determination of the cause and manner of death. Resuscitative artifacts can resemble homicidal or accidental injury and thus possibly be misinterpreted. Occasionally, new technologies and/or medical procedures will create original and/or distinctive artifacts. In 2003, the San Francisco Fire Department emergency personnel began field-testing the Revivant AutoPulse, an automated chest compression device. This device is currently being used in two other counties in the San Francisco Bay Area as well as regions of Florida, Virginia, and Ohio. We present three cases of resuscitative artifact that could be potentially confused with homicidal or accidental injury. These cases illustrate resuscitative artifacts, specifically lateral chest and horizontally oriented upper abdomen cutaneous abrasions created by this automated chest compression device.

Distinguishing Amphetamine, Methamphetamine and 3,4-Methylenedioxymethamphetamine from Other Sympathomimetic Amines After Rapid Derivatization with Propyl Chloroformate and Analysis by Gas Chromatography—Chemical Ionization Mass Spectrometry

Misidentification of ephedrine and pseudoephedrine as methamphetamine has been reported because of similar retention times of their derivatives in gas chromatography as well as very similar mass spectral fragmentation patterns in the conventional electron impact mode of analysis. Recently, a new derivatization of amphetamine and methamphetamine has been described using propyl chloroformate. The derivatization is easily accomplished at room temperature by adding the derivatizing reagent in the extraction solvent because the reagent is stable in the presence of water. The electron impact mass spectrum of derivatized methamphetamine (base peak, m/z 144, other peaks at m/z 102, 58) is similar to the electron impact mass spectrum of both derivatized pseudoephedrine (base peak, m/z 144, other peaks, m/z 102, 58), and ephedrine (base peak, m/z 144, other peaks, m/z 102, 58). Therefore, misidentification of ephedrine and pseudoephedrine as methamphetamine is possible even if this new derivatization technique is used with conventional gas chromatography/electron impact mass spectrometry. We demonstrated that by using chemical ionization mass spectrometry, this problem can be eliminated. In the chemical ionization, using methane as a reagent gas, derivatized methamphetamine showed a protonated molecular ion as a base peak at m/z 236 and other strong peaks at m/z 144 and 119, both derivatized ephedrine and pseudoephedrine showed a base peak at m/z 192 and another strong peak at m/z 148, thus differentiating them clearly from methamphetamine. Amphetamine also showed a protonated molecular ion at m/z 222 and other strong peaks at m/z 130 and 119, whereas phenylpropanolamine after derivatization with propyl chloroformate showed a base peak at m/z 220 and another strong peak at m/z 238, thus differentiating it from amphetamine. The designer drug 3,4-methylenedioxymethamphetamine (MDMA) showed a molecular ion at m/z 279 using electron impact, after derivatization with propyl chloroformate. Using chemical ionization, a relatively stronger protonated molecular ion at m/z 280 was observed. We conclude that using chemical ionization instead of conventional electron impact and propyl chloroformate derivatization, misidentification of ephedrine or pseudoephedrine as methamphetamine or phenylpropanolamine as amphetamine can be eliminated.

A Novel Derivatization of Phenol after Extraction from Human Serum Using Perfluorooctanoyl Chloride for Gas Chromatography-Mass Spectrometric Confirmation and Quantification

Phenol (carbolic acid) is widely used as a disinfectant as well as in the chemical industry as an intermediate in the synthesis of a variety of chemicals. Phenol is also the major metabolite of benzene which is used in many commercial solvents. Phenol is toxic and caustic and may cause death even from dermal absorption. Therefore, measurement of phenol in postmortem blood is essential. The concentration of phenol in blood can be measured by gas chromatography with flame ionization or mass spectrometry. Phenol can also be analyzed by high performance liquid chromatography. However, in forensic toxicology, unambiguous confirmation of phenol by mass spectrometry is as important as quantification in blood. Here we describe a novel derivatization of phenol after extraction with chloroform from human serum using perfluorooctanoyl chloride. The perfluorooctanoyl derivative of phenol showed a strong molecular ion at m/z 490 (relative abundance: 23%) whereas the base peak was observed at m/z 77. The derivative of the internal standard 3,4-dimethylphenol showed a very strong molecular ion at m/z 518 (relative abundance: 56%) and the base peak was observed as m/z 121. The derivative of p-cresol, a chemically related phenolic compound, showed a strong molecular ion at 504 m/z (relative abundance: 54%) and a base peak at m/z 107. We observed baseline separation between derivatized phenol (retention time: 6.1 min), p-cresol (retention time: 7.8 min), and the internal standard (retention time: 9.4 min). We observed no interferences in our assay from grossly hemolyzed serum. Within and between run precision was studied using a serum standard containing 25 mg/L of phenol. The within run precision was 6.6% (mean = 24.3, SD = 1.6 mg/L, n = 8) whereas the between run precision was 8.6% (mean = 25.5, SD = 2.2 mg/L, n = 8). The assay was linear for serum phenol concentrations of 10-200 mg/L. The detection limit was 1 mg/L of serum phenol concentration. The average recoveries were 92.1% to 94.0% for various serum phenol concentrations.

Gas chromatography-electron ionization and chemical ionization mass spectrometric analysis of urinary phenmetrazine after derivatization with 4-carbethoxyhexafluorobutyryl chloride--a new derivative.

Phenmetrazine is a central nervous system stimulant currently used as an anorectic agent. The drug is abused and is reported to cause death from overdose. We describe a new derivatization method for phenmetrazine using 4-carbethoxyhexafluorobutyryl chloride. Quantitation of urinary phenmetrazine can be easily achieved by using N-ethyl amphetamine as an internal standard. The electron ionization mass spectrum of 4-carbethoxyhexafluorobutyryl derivative of phenmetrazine showed a molecular ion at m/z 427 and a base peak at m/z 70. In the methane chemical ionization mass spectrum, the base peak was observed at m/z 428 (protonated molecular ion). In the electron ionization mass spectrum of 4-carbethoxyhexafluorobutyryl derivative of the internal standard, N-ethyl amphetamine we did not observe a molecular ion. However, in the chemical ionization mass spectrum, the protonated molecular ion at m/z 414 was the base peak. The retention time of derivatized phenmetrazine (8.4 min) was substantially longer than the retention time of the underivatized molecule. Moreover, underivatized phenmetrazine showed poor peak shape (substantial tailing) while derivatized phenmetrazine had excellent chromatographic properties. The within-run and between-run precisions of the assay were 2.6% and 3.1% respectively at a urinary phenmetrazine concentration of 10 micrograms/mL. The assay was linear for urinary phenmetrazine concentration of 1 to 100 micrograms/mL with a detection limit of 0.2 microgram/mL.

POSTMORTEM LIPID LEVELS FOR THE ANALYSIS OF RISK FACTORS OF SUDDEN DEATH :USEFULNESS OF THE EKTACHEM AND MONARCH ANALYZERS

Elevated serum cholesterol, triglyceride, and free fatty acid levels have been identified as risk factors for sudden death from cardiovascular disease and increased risk for myocardial ischemia or arrhythmias; therefore, correlation of antemortem and postmortem lipid levels may be useful in establishing the cause, pathophysiology, or familial risk factors of sudden death. In the present study, antemortem (within 72 h) and postmortem (within 24 h) cholesterol, triglyceride, free fatty acid, and albumin levels were analyzed in seven autopsied hospitalized patients from the University of New Mexico Hospital in Albuquerque, New Mexico. The cholesterol, triglyceride, and albumin levels were measured by dry-slide technology on an Ektachem 700 analyzer, and the free fatty acid levels were measured on a Monarch analyzer with a commercially available kit from Wako Chemical. Postmortem cholesterol levels averaged 13% lower than antemortem levels, postmortem triglyceride levels averaged 38% higher than antemortem levels, postmortem free fatty acid levels averaged 23% lower than antemortem levels, and postmortem albumin levels were essentially unchanged (<0.01% higher) from antemortem levels. Whether the antemortem and postmortem differences in lipid levels were the result of postmortem degradation products, a general phenomenon (such as variable enzyme degradation), or an idiosyncracy of the Ektachem or Monarch systems could not be definitely established. These preliminary results suggest that caution should be exercised when interpreting postmortem cholesterol, triglyceride, and free fatty acid levels analyzed on the Ektachem or Monarch systems.

Vertebral artery dissection complicating occipital injection of heparin for treatment of thoracic outlet syndrome.

A 38-year-old woman with a 2-year history of chronic neck pain radiating down her right arm underwent radiological and neurological evaluations, which revealed no anatomical cause for her pain. She sought alternative therapies including intramuscular heparin injections. Following a right occipital injection of heparin, cyanocobalamin, and lidocaine, she had a sudden cardiorespiratory arrest and was successfully resuscitated, but did not regain consciousness.Computed tomography of the head and neck and subsequent autopsy revealed a right vertebral artery dissection, but at autopsy, no significant subarachnoid hemorrhage was noted at the base of the brain. This is the first case report where heparin (a potent anticoagulant) used in an occipital injection was documented to cause a vertebral artery dissection. It is also the first reported case where radiographically and histologically documented vertebral artery dissection did not present with overwhelming subarachnoid hemorrhage at the base of the brain. The subtle gross anatomical findings in this case highlight the importance of evaluating the cervical spinal cord in any case of sudden cardiorespiratory arrest following even apparently minor neck injury.