Biology subjects

Carpenter, R. E.

Publications and source records attributed to Carpenter, R. E..

4 recordsLinked to original sources

bioRxiv2022

Analysis of D- and L- Isomers of (Meth)amphetamine in Human K2EDTA Plasma

Methamphetamine and its metabolite amphetamine are frequently abused drugs. Whether obtained legally or from clandestine laboratories it is of relevance to determine the chiral makeup of these drugs for investigative purpose. Although urine and oral fluid matrices are commonly offered, less available to independent laboratories are techniques to verify dextro (D-) or levo (L-) (meth)amphetamine from human K2EDTA plasma. This paper outlines the development and validation of a method that includes the addition of internal standard and a two-step liquid-liquid extraction to remove the analytes from human K2EDTA plasma by triple quadrupole mass spectrometry (LC-MS/MS). The assay was validated according to the United States Food and Drug Administration and College of American Pathologists guidelines, including assessment of the following parameters in plasma validation samples: linear range, limit of detection, lower limit of quantitation, matrix effects, inter- and intra-day assay precision and accuracy, carry over, linearity of dilution, matrix effects and stability. The outcome is a validated and reliable method for the determination of D- and L-isomer concentration of meth(amphetamine) human plasma samples that can be easily adopted by independent clinical laboratories.

biochemistry↗

bioRxiv2022

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

A decentralized surveillance system to identify local outbreaks and monitor SARS-CoV-2 Variants of Concern is one of the primary strategies for the pandemics containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the technology is still cost-prohibitive for decentralized sequencing, particularly in small independent labs with limited resources. We have optimized the Illumina COVID-seq protocol to reduce cost without compromising accuracy. 90% of genomic coverage was achieved for 142/153 samples analyzed in this study. The lineage was correctly assigned to all samples (152/153) except for one. This modified protocol can help laboratories with constrained resources contribute to decentralized SARS-CoV-2 surveillance in the post-vaccination era.

genomics↗

bioRxiv2022

Quantifying 64 drugs, illicit substances, and D- and L- isomers in human oral fluid with liquid-liquid extraction

Although human oral fluid has become more routine for quantitative drug detection in pain management, detecting a large scope of medications and substances is costly and technically challenging for laboratories. This paper presents a quantitative assay for 64 pain medications, illicit substances, and drug metabolites in human oral fluid. The novelty of this assay is that it was developed on an older model AB SCIEX 4000 instrument and renders obscure the need for more technical and expensive laboratory equipment. This method includes addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water and separation and detection was accomplished using triple quadrupole mass spectrometry (LC-MS/MS). Separation was achieved using reverse-phase liquid chromatography with detection by LC-MS/MS. A second injection was done in negative mode to determine THC-COOH concentration as an indicator of THC. An aliquot of the (already) extracted samples was analyzed for D- and L- isomers of amphetamine and methamphetamine using a chiral column. The standard curve spanned from 5 to 2000 ng/mL for most of the analytes (1 to 2000 ng/mL for fentanyl and THC-COOH) and up to 1000 ng/mL for 13 analytes. Pregabalin and gabapentin ranged from 25 to 2000 ng/mL. The result is a low-cost method for the sensitive detection of a wide-ranging oral fluid menu for pain management. This assay has a high sensitivity, and good precision and accuracy for all analytes with an older model mass spectrometer.

biochemistry↗

bioRxiv2022

Confirming Multiplex Q-PCR Use in COVID-19 with Next Generation Sequencing: Strategies for Epidemiological Advantage

Rapid classification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the Variants of Concern and tracking this pandemic. However, NGS remains expensive and time-consuming for large-scale monitoring of COVID-19. This study analyzed a total of 78 de-identified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole genome sequencing then compared with two commercially available Q-PCR assays for spike protein mutation(s). The data showed good concordance with Q-PCR and NGS analysis for specific SARS-COV-2 lineages and characteristic mutations. Deployment of Q-PCR testing to detect known SARS-COV-2 variants may be extremely beneficial. These assays are quick and cost-effective, thus can be implemented as an alternative to sequencing for screening known mutations of SARS-COV-2 for clinical and epidemiological interest. The findings support the great potential for Q-PCR to be an effective strategy offering several COVID-19 epidemiological advantages.

genomics↗