David W. Blank

Development and optimization of simplified LC–MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE)

25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC-MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid-liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC-MS/MS technique permits the measurement of both 25-hydroxyvitamin D(2) (25-OH D(2)) and the 25-hydroxyvitamin D(3) (25-OH D(3)) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 μM column with a 2.1 mm × 20 mm C18 3.5 μM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D(3) and 25-OH D(2).The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with either pure triple stripped blank serum or diluted in 6% phosphate buffer saline at pH 7.2. Linearity was tested up to 160 nmol/L for 25-OH D(3) and 75 nmol/L for 25-OH D(2). Low limit of quantification (LLOQ) were established at 3 nmol/L for 25-OH D(2) and 4 nmol/L for 25-OH D(3). Inter-assay and intra-assay precision (CV%) was determined using 3 levels of commercial controls (Utak, CA, USA) for 25-OH D(2) and 25-OH D(3). Results obtained for intra-assay and inter-assay precision (CV%) were 1.1-3.4% and 5-8.9% respectively for the PPE/centrifugation technique and 2.0-3.1% and 4.6-6.6% for the PPE/filtration technique. Accuracy was estimated with the same commercial controls: % bias was -11.2 to 4.9% with PPE/centrifugation and -3.2 to 6.1% with PPE/filtration. 25-OH D(2) and 25-OH D(3) concentrations in human serum with LLE were compared to the new extraction methods using either PPE/centrifugation or PPE/filtration. Correlations comparing the two methods revealed a slope approximately 1.0±0.3 with R≥0.98 with a bias<1 nmol/L. In summary, the new LC-MS/MS method described in this report using an on-line SPE technique with a simple off-line pre-treatment is faster, cost-effective, more reliable and more robust than current and widely used LLE/centrifugation methods coupled with LC-MS/MS.

Analytical interferences resulting from intravenous lipid emulsion

Context. Lipid resuscitation therapy using intravenous lipid emulsion (IVLE) for drug overdoses has gained widespread use. However, there is little information regarding its adverse effects. Objectives. We performed lipemic interference studies on typical automated platforms to investigate the potential of lipid resuscitation therapy to interfere with the reliability and turnaround time of analytes that would be of interest in acute intoxications. We also tested methods to minimize interferences. Materials and methods. Serum pools were supplemented with increasing concentrations of Intralipid-20%® (0–30%). Analyses were performed on Beckman-Coulter DXC800 and DXI and Roche Modular-P. Analytes demonstrating significant interference were re-measured after centrifugation (14 000 × g for 10 minutes). Results. Triglyceride and glycerol-blanked triglyceride concentrations were similar in IVLE-free samples. However, with addition of IVLE, concentrations were markedly different (139 vs. 76 mmol/L). There was no appreciable interference on the troponin-I, sodium, potassium, chloride, calcium, bicarbonate or urea assays. Albumin and magnesium assays demonstrated significant interference. Amylase, lipase, phosphate, creatinine, total protein, ALT, CK and bilirubin became unmeasurable in IVLE-supplemented samples. Whereas glucose measurement by potentiometry was free of interference, colorimetric methodology was error prone. Centrifugation removed > 90% of glycerol-blanked triglyceride (max = 5.8 mmol/L), dramatically reducing lipid interferences. Discussion. IVLE results in appreciable analytical interferences at concentrations demonstrated in lipid resuscitation therapy. Of particular concern is the marked interference on glucose and magnesium, which may result in unsuccessful and potentially harmful interventions. Major implications for patient care include reporting of incorrect results and delays in the reporting of time-sensitive results. Whenever possible, blood samples should be collected prior to initiating lipid therapy. Interferences can be minimized by brief centrifugation at relatively low speeds on equipment readily available in most core labs.

Maternal predictors of RBC folate levels in an urban Canadian population

OBJECTIVE To assess factors associated with low Red Blood Cell folate (RBCf) levels in an obstetric population in a tertiary centre. METHODS Cross-sectional study. Three hundred and fifty women completed a questionnaire detailing use of folic acid supplementation, and had their RBCf levels measured. Values ≥ 906 nmol/L were considered optimal. Factors associated with optimal RBCf were assessed, individually and in a logistic regression model. RESULTS Median RBCf was 1282 nmol/L. Thirty-five women (10%) had suboptimal levels. Predictors of suboptimal RBCf were non-Caucasian ethnicity, non-consumption of folic acid supplementations, and inadequate health care provider information regarding the benefits of folic acid consumption. CONCLUSION Although, in our population, a high proportion of women achieved optimal levels of RBCf, some women remain at risk due to inadequate folate consumption. Patient and health care provider education regarding folate can still be improved, particularly in the groups identified to be at greater risk.

Comment on complications following antidotal use of intravenous lipid emulsion therapy (Levine et Al., J Med Toxicol 2013).

A published case series of Levine et al. reporting complications following antidotal use of intravenous lipid emulsion (ILE) is a well-written and much needed critical analysis [1]. In addition to the clinical complications of acute pancreatitis and respiratory distress syndrome, the authors also address the analytical complications of lipemic interference. ILE interferences can lead to delayed reporting of time-sensitive results with dire consequences as demonstrated by the loss of a potential organ donor candidate. Additionally, erroneous results may be used to guide therapy [2].

Investigating the optimal gammaglobulin concentration cut-off for serum immunofixation electrophoresis in hypogammaglobulinemic patients.

A previous study showed that hypogammaglobulinemia without a monoclonal (M) peak on serum protein electrophoresis (SPE) was present in 8% of multiple myeloma patients [1]. SPE and immuno fixation electrophoresis (IFE) are major tools for screening and for the diagnosis ofmonoclonal gammopathies includingmultiplemyeloma [2,3]. The IFE positive rate in two hypogammaglobulinemic cohorts was 9.7% and12% [4,5]with gammaglobulin cut-offs of 7.0 and 5.5 g/L respectively. It is common laboratory practice to perform IFE as a reflex test when anomalies such as hypogammaglobulinemia are identified on SPE, but no consensus exists on the gammaglobulin concentration cut-off [6]. Indeed different clinical laboratories use widely different gammaglobulin concentrations for reflex IFE and these values are often well below the lower limit of the gammaglobulin reference range. There is no study found in the literature to address whether a lower gammaglobulin cut-off can increase IFE positive rates without a significant loss of sensitivity and therefore reduce the use of IFE. We carried out a retrospective study at the Royal Victoria Hospital of the McGill University Health Center on all new hypogammaglobulinemic patients without any suspicious peaks on SPE from January 2005 to February 2011. We identified 194 patients with a gammaglobulin concentration of 6.2 g/L or below based on the 2.5th percentile of our own normal patient population. The first SPE and the first IFE results using the same serum were used for the study. All 194 SPEs were reviewed so that no suspicious peaks, no asymmetry in the β1 and β2 regions and no restriction in the γ region were present. Furthermore, we studied age, gender, SPE fractional concentrations (albumin, α1-globulin, α2-globulin, β1-globulin, β2-globulin and γ-globulin) and their ratios (α2/α1, β2/β1 and albumin/gamma), as well as selected laboratory test results (serum total protein, calcium, phosphate, creatinine, lactate dehydrogenase; IgA, IgG, IgM, β2 microglobulin and haemoglobin) as potential predictors of IFE positivity. In addition, the available serum free light chain assay results were examined in 35 patients. Among 194 hypogammaglobulinemic patients, 37 were IFE positive (positive rate of 19.1%) including 4 free κ, 5 free λ and 1 IgA λ plus free λ. The gammaglobulin concentration in the IFE negative group ranged from 0.5 g/L to 6.2 g/L with a median of 4.6 g/L. In the