Development of an improved standard reference material for folate vitamers in human serum.

The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material® (SRM®) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-µg folic acid supplement 1 h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates.

Collaborative Method Performance Study of the Measurement of Nicotine, Its Metabolites, and Total Nicotine Equivalents in Human Urine.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements.Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry.Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results.Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine.Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083-90. ©2018 AACR.

Development of a Cigarette Tobacco Filler Standard Reference Material.

A new tobacco filler Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST) in September 2016 with certified and reference mass fraction values for nicotine, N-nitrosonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and volatiles. The constituents have been determined by multiple analytical methods with measurements at NIST and at the Centers for Disease Control and Prevention, and with confirmatory measurements by commercial laboratories. This effort highlights the development of the first SRM for reduced nicotine and reduced tobacco-specific nitrosamines with certified values for composition.

Influence of chemical straightening on the stability of drugs of abuse in hair.

Chemical straightening, also known as a relaxer, is ubiquitously used among African American women to obtain straighter hair compared with their natural tresses. This study focused on the stability of drugs of abuse in hair after a single application of the relaxer. Commercially available 'Lye' or 'No-Lye' chemical straightening products (Silk Elements™) were applied in vitro to drug-fortified hair (standard reference materials (SRM) 2379 and 2380) and hairs clipped from established drug users. Target analytes (cocaine (COC), benzoylecgonine (BZE), cocaethylene (CE), phencyclidine and tetrahydrocannabinol) were isolated using solid-phase extraction and then analyzed with isotope dilution gas chromatography-mass spectrometry with selective ion monitoring. After either treatment, drug concentrations were significantly (P < 0.05) decreased in both the SRM sample and the hair from authentic abusers. In the SRM groups, 6-67% of the original concentration remained after a single chemical treatment. Similarly, only 5-30% of the original concentration remained in authentic drug hairs that had formerly tested positive for COC, BZE and CE.

Monitoring of in vivo manipulation of nitric oxide synthases at the rat retina using the push-pull perfusion sampling and capillary electrophoresis.

Proteins play a variety of functional roles in tissues that underlie tissue health. The measurement of protein function is important to both understand normal and dysfunctional tissue states. Low-flow push-pull perfusion sampling (LFPS) has been used to collect submicroliter volumes of extracellular fluid which are well suited to capillary electrophoresis for compositional quantitative analysis. In this study, LFPS is used to deliver pharmacological agents to the in vivo retinal tissues at the probe sampling tip during sampling to measure protein function. Two native nitric oxide synthase enzymes were pharmacologically inhibited and the enzyme product NO metabolite, nitrate, was determined with capillary electrophoresis from the perfusates. LFPS delivered inhibitors including the non-selective N(G)-nitro-Larginine methyl ester (L-NAME), the nNOS selective 7-nitroindazole (7-NI), and eNOS N5-(1-imioethyl)-L-ornithine, dihydrochloride (L-NIO) were perfused to the sampling region either directly over a rat retina optic nerve head or 1-mm peripheral to the ONH. At the PONH, 65, 55 and 60% of baseline nitrate levels, respectively, were observed with inhibitor infusion. These are statistically significant (P<0.05) compared to saline drug infusion. However, infusion of the inhibitors to the ONH did lead to significant nitrate concentration decreases. This data suggests that the endogenous enzymes, nNOS and eNOS, are both spatially and functionally localized to the PONH at the in vivo rat retina.

High temporal resolution coupling of low-flow push-pull perfusion to capillary electrophoresis for ascorbate analysis at the rat vitreoretinal interface.

A system is presented demonstrating the high-temporal resolution coupling of low-flow push-pull perfusion sampling (LFPS) to capillary electrophoresis for the absorbance measurement of ascorbate at the rat vitreoretinal interface. This system holds all separation components at a low pressure as the means for withdrawing sample during LFPS. The system uses a flow-gated interface to directly couple the withdrawal capillary from the LFPS probe to a separation capillary and eliminates the need for any offline sample handling. The temporal resolution of the system was limited by injection time and is less than 16 s. This high temporal resolution was applied to the monitoring of in vivo ascorbate levels at the rat vitreoretinal interface. Baseline concentrations of ascorbate were found to be 86 microM +/- 18 microM at the vitreoretinal interface. Baseline concentrations matched well with those obtained for the postmortem bulk vitreous analysis. Upon stimulation with 145 mM K(+), a maximum increase in baseline values between 32-107% for n = 3 was observed. This system demonstrates the first in vivo temporal study of ascorbate at the rat vitreoretinal interface.

Measurement of region-specific nitrate levels of the posterior chamber of the rat eye using low-flow push-pull perfusion.

The determination of the presence of nitric oxide metabolites in the rat vitreous cavity in a regioselective manner is complicated by the size and shape of the eye as well as the diffusivity of the molecule and its metabolites. In this work, in vivo low-flow push-pull perfusion sampling was utilized with a rapid capillary electrophoretic assay to monitor levels of the major NO metabolite, nitrate, at the vitreoretinal interface (VRI) of normal and aged rat models. The sampling probe tips were placed in three different positions in the posterior chamber through a 29-gauge guide needle. Sampling was performed along the VRI over the optic nerve head and regions peripheral to the optic nerve head. Additionally, samples were collected from the middle vitreous region to compare to VRI sampling. A significant (P < 0.05) difference in the perfusate nitrate concentration was observed in each location, which may be due to the source of NO production or the clearance mechanism of the molecule from the vitreous cavity. Infusion of L-NAME with physiological saline led to a significant decrease (35%) in the observed nitrate level. LFPPP was then utilized to observe nitrate levels after an average of 4.5 months of aging. A 3-fold increase in the mean level of nitrate over the optic nerve head was observed in mature animals compared to younger control animals. Precise measurement of NO metabolites along the VRI may provide insights into the function of NO in maintaining homeostatic conditions and the molecular changes at the diseased retina.