Strategy and validation of a consistent and reproducible nucleic acid technique for mycoplasma detection in advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are required to maintain their quality and safety throughout the production cycle, and they must be free of microbial contaminations. Among them, mycoplasma contaminations are difficult to detect and undesirable in ATMP, especially for immunosuppressed patients. Mycoplasma detection tests suggested by European Pharmacopoeia are the "culture method" and "indicator cell culture method" which, despite their effectiveness, are time consuming and laborious. Alternative methods are accepted, provided they are adequate and their results are comparable with those of the standard methods. To validate a novel in-house method, we performed and optimized, a real time PCR protocol, using a commercial kit and an automatic extraction system, in which we tested different volumes of matrix, maximizing the detection sensitivity. The results were compared with those obtained with the gold standard methods. From a volume of 10 ml, we were able to recognize all the mycoplasmas specified by the European Pharmacopoeia, defined as genomic copies per colony forming unit ratio (GC/CFU). Our strategy allows to achieve faster and reproducible results when compared with conventional methods and meets the sensitivity and robustness criteria required for an alternative approach to mycoplasmas detection for in-process and product-release testing of ATMP.

Comparison of two methods for the extraction of ethylglucuronide from hair.

The aim was the comparison between the Society of Hair Testing (SoHT) consensus for the use of alcohol markers which powdering hair for the extraction of ethylglucuronide (EtG) in water and extraction using the patented M3 Reagent Test kit on cut hair. Hair samples were cut into small segments and washed twice with methanol and diethyl ether. The SoHT-Consensus entails the extraction of pulverised hair in water. This is obtained by incubation of 25 mg of hair at room temperature overnight and 2 h sonication, even if the overnight incubation is not mandatory. The M3 method entails incubation of 25 mg of cut hair with the M3-Reagent at 100°C for 60 min. After centrifugation, the supernatant is injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples (191) were collected in the APSS laboratory in Trento, Italy, between 2021 and 2022. The limit of quantification (LOQ) was set at 5 pg/mg for the pulverised and M3-Reagent methods. Assays showed good linearity above the range of LOQ-300 pg/mg. Precision (within 20%) values were also obtained using both methods. In the Passing-Bablock linear regression, the final regression curve between M3 (y) and the pulverising method (x) showed good agreement; the Bland-Altman analysis did not show any significant bias between the two methods. The M3-Reagent method, due to cut hair use, is easy to perform, saves time and allows for a smaller sample quantity loss with use of nondisposable grinding jars for the ball mill to obtain the extraction of EtG.

An LC-MS/MS method for the determination of drugs of abuse included THC-COOH, EtG, and NPS, using a single hair extraction sample.

Hair analysis plays an important role in the determination of drugs of abuse in both forensic and clinical toxicology investigations. The analysis of different substances often requires the use of different sample preparation methods, thereby increasing the amount of hair sample and time required. In the present study, a fast method involving a combination of a single 25 mg hair extraction procedure and four liquid chromatography-tandem mass spectrometry methods using the same chromatographic phases and column was developed and validated. The target was the identification and quantification of various commonly abused drugs and their metabolites, including amphetamines, cocaine, opioids, cannabinoids, THC-COOH and EtG, and more than 140 new psychoactive substances, including synthetic cannabinoids, phenethylamines, synthetic opioids, methylphenidate, cathinone, piperidine, and tryptamines.