Vitamin D levels and relative insufficiency in Saskatchewan.

OBJECTIVE: To determine the extent of vitamin D3 deficiency and levels in pregnant First Nations and non-First Nations women in SK. Also, to determine the distribution of vitamin D3 values in the general population in SK. METHODS: Vitamin D3 levels were measured by LC-MS/MS from 19,181 consecutive patient blood/serum samples received at the Saskatchewan Disease Control Laboratory, and from 743 First Nations, and 301 non-First Nations pregnant women in SK. RESULTS: The ages of the 19,181 patient samples ranged from day 1 (0 years) to 102 years. Of the total, 14,658 were female, and 4523 were males. 30.8% had relative vitamin D3 insufficiency (50-75 nmol/L), and 22.5% were in the deficient range (<50 nmol/L). In summer, a larger percentage of SK patients are in the optimum range, whereas in winter, the number of patients in the vitamin D3 deficiency range increased to 33.0% from 14.1%. Samples from pregnant women were collected during the first trimester of pregnancy. Whereas non-First Nations pregnant women had similar vitamin D3 levels to non-pregnant women in SK, vitamin D3 levels were significantly lower than the optimum of 75 nmol/L in pregnant First Nations women than in non-First Nations women. 29.7% of First Nations pregnant women were in the relative insufficiency range, and 45.6% were vitamin D3 deficient. CONCLUSIONS: First Nations pregnant women have lower vitamin D3 levels than non-First Nations pregnant women. This puts them and their unborn babies at high risk of a diverse range of disorders associated with vitamin D3 deficiency or insufficiency.

LC-MS/MS techniques for high-volume screening of drugs of abuse and target drug quantitation in urine/blood matrices.

Liquid chromatography-tandem mass spectrometry, employing electrospray ionization (ESI), has been applied in the analysis of many drugs and drug metabolites. Sample preparation has been an important part of this technique when analyzing biological samples. Here we describe a high-volume urine screening technique for approximately 40 different drugs of abuse as well as methods for quantification of many other drugs in serum, plasma, and whole blood. These techniques can be used in many different settings from clinical and forensic toxicology examinations to pharmacokinetic studies. Sample preparation procedures range from simple "dilute and shoot" methods to more extensive solid-phase extraction techniques.

Opiate screening and quantitation in urine/blood matrices using LC-MS/MS techniques.

Here we describe a high-volume urinary screening technique for opiate drugs as well as other narcotic analgesics. We also describe methods for quantification of the same drug species in serum, plasma, and whole blood. Screening and quantitation of these types of drugs have presented many challenges, among them the potentially low levels in both abuse and therapeutic situations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing electrospray ionization (ESI), has been able to provide the sensitivity needed for the analysis of many drugs and metabolites. These techniques can be used in many different settings from clinical and forensic toxicology examinations to pharmacokinetic studies and, with appropriate considerations, be applied to different sample matrices. Sample preparation procedures range from simple "dilute and shoot" methods to more extensive solid-phase extraction techniques.

Elevated neonatal 3-OH isovalerylcarnitine due to breast milk sources in maternal 3-MCC deficiency.

We report a positive newborn screen for 3-hydroxyisovalerylcarnitine (C(5)OH) with an absence of 3-methylcrotonyl-coenzyme A carboxylase deficiency in the neonate. Subsequent blood tests demonstrated persistently elevated C(5)OH. Serial testing of the mother identified markedly elevated C(5)OH in both maternal blood and breast milk. High C(5)OH milk concentrations provide a significant source of C(5)OH to the nursing neonate and possibly explains its persistent elevation in the neonate, a commonly observed finding in maternal 3-MCC deficiency.

Liquid chromatography with post-column reagent addition of ammonia in methanol coupled to negative ion electrospray ionization tandem mass spectrometry for determination of phenoxyacid herbicides and their degradation products in surface water.

A new liquid chromatography (LC)-negative ion electrospray ionization (ESI(-))-tandem mass spectrometry (MS/MS) method with post-column addition of ammonia in methanol has been developed for the analysis of acid herbicides: 2,4-dichlorophenoxy acetic acid, 4-chloro-o-tolyloxyacetic acid, 2-(2-methyl-4-chlorophenoxy)butyric acid, mecoprop, dichlorprop, 4-(2,4-dichlorophenoxy) butyric acid, 2,4,5-trichlorophenoxy propionic acid, dicamba and bromoxynil, along with their degradation products: 4-chloro-2-methylphenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol and 3,5-dibromo-4-hydroxybenzoic acid. The samples were extracted from the surface water matrix using solid-phase extraction (SPE) with a polymeric sorbent and analyzed with LC ESI(-) with selected reaction monitoring (SRM) using a three-point confirmation approach. Chromatography was performed on a Zorbax Eclipse XDB-C18 (50 x 4.6 mm i.d., 1.8 mum) with a gradient elution using water-methanol with 2 mM ammonium acetate mobile phase at a flow rate of 0.15 mL/min. Ammonia in methanol (0.8 M) was added post-column at a flow rate of 0.05 mL/min to enhance ionization of the degradation products in the MS source. One SRM transition was used for quantitative analysis while the second SRM along with the ratio of SRM1/SRM2 within the relative standard deviation determined by standards for each individual pesticide and retention time match were used for confirmation. The standard deviation of ratio of SRM1/SRM2 obtained from standards run on the day of analysis for different phenoxyacid herbicides ranged from 3.9 to 18.5%. Limits of detection (LOD) were between 1 and 15 ng L(-1) and method detection limits (MDL) with strict criteria requiring <25% deviation of peak area from best-fit line for both SRM1 and SRM2 ranged from 5 to 10 ng L(-1) for acid ingredients (except dicamba at 30 ng L(-1)) and from 2 to 30 ng L(-1) for degradation products. The SPE-LC-ESI(-) MS/MS method permitted low nanogram-per-liter determination of pesticides and degradation products for surface water samples.

Drugs of abuse testing by tandem mass spectrometry: a rapid, simple method to replace immunoassays.

PRIMARY OBJECTIVE: To replace immunoassay screening for drugs of abuse (DOA) with a cost-effective tandem mass spectrometry method. SECONDARY OBJECTIVE: To substantially expand the drugs of abuse assay menu. DESIGN AND METHODS: The requirement was to perform high throughput DOA screening for 200 urine specimens/day for 40 drugs/metabolites. The total analysis time had to be <5 min. We used UPLC chromatography, small particle size LC columns and fast scanning tandem mass spectrometry. Urine samples were hydrolyzed enzymatically, diluted and injected with isotopically labeled internal standards. The data produced was transferred by exporting reports as text files to a LIMS system followed by auto certification of the results. RESULTS: 40 different drugs were separated by UPLC (ultra pressure liquid chromatography) with a run time of 5.2 min. Detection limits were below our cut-off values. Individual drug species instead of drug classes were identified; correlation with GC/MS was excellent. A high throughput, robust assay with acceptable accuracy, precision and specificity was developed. The procedure can also be used as a quantitative method with simple modifications. CONCLUSIONS: An improved, high throughput, cost-effective method for drugs of abuse screening has been implemented. GC/MS confirmations were reduced or eliminated. The new procedure is a viable alternative to our previous immunoassay method. Acceptable turn around times, an expanded menu, simplified sample preparation and analytical reliability makes this method a desirable option in the clinical laboratory setting.

Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method.

17alpha-Hydroxyprogesterone is a metabolic precursor of cortisol; elevated levels of 17alpha-hydroxyprogesterone are indicative of congenital adrenal hyperplasia. Traditional determination by immunoassay is plagued by poor antibody specificity, resulting in significant interferences. This study explores an LC-MS/MS method for the quantitation of 17OHP in serum. Deuterated 17alpha-hydroxyprogesterone was added as internal standard, followed by solid-phase extraction, HPLC separation with a C16-amide reverse-phase column with run time of 7 min, and quantification by MS/MS (positive electrospray ionisation) in the selected reaction monitoring mode (SRM). Transitions monitored were 331>109 for the analyte and 339>113 for the deuterated internal standard. Intra-assay precision (%R.S.D.) was 7.4% at 7 nmol/L, inter-assay precision (%R.S.D.) at 2, 7 and 27 nmol/L was 15.4, 10.0 and 7.9% and accuracy at 0.9 nmol/L was 100%. The method was linear from 0.156 to 80 nmol/L. Lower limit of quantitation was 0.2 nmol/L, providing meaningful data for patients within normal range as well as those with elevated levels.