Gary L. Lensmeyer

The C-3 Epimer of 25-Hydroxyvitamin D3 Is Present in Adult Serum

CONTEXT Epimers have identical molecular structure but differ in stereochemical configuration. It is widely believed that the C-3 epimer of 25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)] is found only in neonates. However, this epimer was recently detected in a limited number of adults. The physiological importance of 3-epi-25(OH)D(3) is uncertain but might affect 25-hydroxyvitamin D test results and thereby reliability of the 25-hydroxyvitamin D(3) [25(OH)D(3)] measurement. OBJECTIVE This project describes development of a highly sensitive method for 3-epi-25(OH)D(3) measurement and establishes the prevalence of this epimer in adult clinical serum specimens. DESIGN, SETTING, PARTICIPANTS, AND MAIN OUTCOME MEASURE: Serum 25(OH)D(3), 3-epi-25(OH)D(3), and 25(OH)D(2) concentrations were determined in a cohort of patients (n = 214; age neonate to 80+ yr). High-performance liquid chromatography with ultraviolet detection and high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization equipped with cyanopropyl analytical columns were used to baseline separate and quantitate 25(OH)D(3), 3 epi-25(OH)D(3), and 25(OH)D(2). RESULTS The C-3 epimer was detected in 212 of 214 (99%) of samples. Concentrations ranged from 1 to 93 ng/ml for 25(OH)D(3) and 0.1 to 23.7 ng/ml for 3-epi-25(OH)D(3). The relative amounts of epimer to 25(OH)D(3) ranged from 0 to 25.5% (mean 4.75%). The epimer amount increased as 25(OH)D(3) increased in a nonlinear mode. In sera with approximately the same 25(OH)D(3) concentration, the ratio of epimer to 25(OH)D(3) varied, e.g. at 25(OH)D(3) values of 20-22 ng/ml, the ratio varied from 2-8.5%. CONCLUSION 3-Epi-25(OH)D(3) is present in the majority of human serum specimens. Although this concentration is generally low, further work must investigate the impact of 3-epi-25(OH)D(3) on the various 25-hydroxyvitamin D assays and ultimately what information, if any, C-3 epimer measurement can provide clinically.

NHANES Monitoring of Serum 25-Hydroxyvitamin D: A Roundtable Summary

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later.

Optimized high-performance liquid chromatographic method for determination of lamotrigine in serum with concomitant determination of phenytoin, carbamazepine, and carbamazepine epoxide.

Lamotrigine (LG), phenytoin (PY), carbamazepine (CM), and carbamazepine epoxide (CE) are measured with an optimized procedure that uses thin sorbent extraction disks and a highly selective, sterically protected bonded silica high-performance liquid chromatography (HPLC) column. Routinely, serum (200 microliters at pH 6.8 with cyheptamide as internal standard) is applied to an Empore octyl (C8) solid-phase extraction disk to isolate the drugs. a water wash removes interferences, and the retained drugs are eluted with a small volume of solvent. The eluate is directly injected onto a Zorbax Stable Bond cyanopropyl HPLC column with quantification at 214 nm. Evaporation-concentration steps are unnecessary. Overall, for all drugs, between-run precision coefficients of variation (n = 16 each) ranged from 2.1% to 4.9% at concentrations from 0.75 to 20.5 mg/l; extraction recoveries fell within a range of 96% to 110% at concentrations of 2, 10, and 30 mg/l tested for each drug; the lowest limit of detection was 0.15 to 0.35 mg/l. The analytical response was linear for each drug > 80 mg/l (LG) and > 50 mg/l (PY, CM, and CE). Optimization graphs are presented to illustrate the rationale for selection of test parameters for a robust method. In addition, a comparison study between two commercial laboratories demonstrates accuracy problems associated with LG testing.

25-Hydroxyvitamin D Measurement, 2009: A Review for Clinicians

As clinicians are more widely appreciating the endemic nature of low vitamin D status, measurement of serum 25-hydroxyvitamin D (25(OH)D), the accepted measure of vitamin D status, has increased. Challenges to 25(OH)D measurement include the presence of 2 forms of vitamin D-ergocalciferol and cholecalciferol (vitamin D(2) and vitamin D(3), respectively)- and the hydrophobic nature of vitamin D. The current state of 25(OH)D measurement is reviewed; modest differences between methodologies persist and confound the application of a single cut point (e.g., 30 ng/mL/75 nmol/L) for the diagnosis of low vitamin D status. The absence of standard calibrators contributes to between-laboratory differences in 25(OH)D measurement. Until there is improved assay standardization and subsequent evidence-based consensus, it seems premature to recommend widespread screening 25(OH)D measurement. Selectively obtaining 25(OH)D measurement in individuals at clinical risk for vitamin D deficiency and/or those most likely to promptly experience benefits from supplementation seems appropriate.

Optimized method for determination of gabapentin in serum by high-performance liquid chromatography

The anticonvulsant drug gabapentin and its heptaneacetic acid analog—used here as an internal standard—are isolated from serum (pH 9) with an octyldecyl (C-18) solid-phase sorbent column. To enhance analytical detection, trinitrobenzene derivatives of these extracted compounds are prepared quickly within 10 min. To furthur improve chromatographic selectivity, the derivatives are concentrated on a thin C-18 solid-phase membrane and interferences are washed away. The retained purified derivatives are eluted from the membrane with a small volume of solvent and the eluate is directly injected onto an Ultrasphere C-18 high-performance liquid chromatography column with quantification at 340 nm. No evaporation-concentration steps are necessary. Recoveries (extraction) of gabapentin and the internal standard are 94.2 ± 2.9% and 98 ± 2.0%, respectively. Analytical responses are linear from lower limit of sensitivity of 0.05 mg/L up to at least 10 mg/L. Between-run coefficients of variation (CV) range from 2.3 to 2.9% through the concentration range 0.5–4.0 mg/L. To illustrate the rationale for selection of test parameters for a robust method, we present optimization graphs for these processes. Moreover, we discuss the advantage of the packed cartridge and membrane sorbents as companion extraction devices.

Therapeutic monitoring of topiramate: evaluation of the saturable distribution between erythrocytes and plasma of whole blood using an optimized high-pressure liquid chromatography method.

Topiramate (TPM) reportedly binds in a saturable manner to erythrocytes but minimally to plasma proteins. Two studies were performed to evaluate this distribution phenomenon. In all studies, TPM was measured with a newly developed, optimized procedure that uses octyldecyl (C-18) solid phase sorbents disks/packed cartridges and a DB-1 methylsilicone capillary gas chromatography (GC) column. Between-run precision coefficients of variation (CVs) (n = 16) ranged from 3.6%-5.6% at concentrations from 3.0 to 15 microg/mL, with low limit of detection of 0.2 to 0.3 microg/mL. For the distribution studies, drug-free whole-blood specimens from five healthy adult volunteers were supplemented with TPM and used to test the influence of TPM concentration and HCT differences on the plasma/blood (P/B) distribution ratio of TPM. In study A, TPM concentration was varied (1-15 microg/mL) and HCT remained constant (40% +/- 5%). In study B, TPM (3 microg/mL) was added to blood specimens comprising a range of HCT values (20%-40%). Study A results were: mean TPM P/B ratios: 0%, 14.2% +/- 5%, 44.2% +/- 4%, 76% +/- 5.5% at 1, 3, 5, 15 microg/mL, respectively. Data between each group were statistically different (p < 0.001). Study B results were: mean TPM P/B ratio: 17.3% +/- 7.3%, 27.5% +/- 10.1%, 39.8% +/- 8% and 56.1% +/- 8.8% at HCT values of 40%, 32%, 26.5%, 20%, respectively. The TPM P/B ratio was significantly inversely correlated to HCT (r = -905, p < 0.001). TPM P/B partitioning was not temperature-dependent. Researchers concluded that the saturable binding of TPM to RBC is significant and is correlated to HCT. As a result, TPM in the plasma fraction of whole blood will increase when HCT decreases and as total TPM concentration in whole blood increases.

Use of particle-loaded membranes to extract steroids for high-performance liquid chromatographic analyses improved analyte stability and detection

Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using the novel particle-loaded octyl (C8)-bonded silica in PTFE membrane. Extracts are directly injected, without further concentration, onto a narrow (2.0 mm) or conventional (4.6 mm) bore octyldecyl (C18) HPLC column. Method performance data demonstrate linearity from 0.4 microgram/dl (low limit of detection) up to at least 60 micrograms/dl. Extraction recoveries exceeded 85% and precision (between-run) R.S.D.s averaged < 5%. Interferences were minimal and selectivity was improved over conventional immunochemical steroid assays. When compared to large particle sorbents packed in columns or to traditional liquid-liquid extractions, the membrane extracted steroids in less time, used less reagent, and had smaller elution volumes, thereby obviating steroid instability/adsorption problems associated with traditional concentrating techniques required to improve analytical sensitivity.

Lamotrigine pharmacokinetics in patients receiving felbamate

Drug interactions can significantly complicate the management of patients receiving multiple medications. It is essential therefore that potential pharmcokinetic interactions be evaluated as new antiepileptic medications are introduced. Lamotrigine (LTG) is a recently marketed medication whose pharmacokinetics are significantly influenced by concomitant drugs. Felbamate (FBM), another relatively new antiepileptic agent has been associated with multiple interactions including both enzyme induction and inhibition. The purpose of the present pilot study was to evaluate potential differences in lamotrigine kinetics in six patients concomitantly receiving FBM compared to five patients receiving lamotrigine as monotherapy. There was no statistically significant differences in either apparent LTG oral clearance (0.026 +/- 0.005 vs. 0.024 +/- 0.01 l/kg per h, respectively), or in mean elimination half-life (33.7 +/- 7.5 vs. 40.2 +/- 15.05 h, respectively). Oral clearance values in our patients are also consistent with data reported previously in the literature. Data from this pilot study suggest that a marked effect of FBM upon lamotrigine pharmacokinetics is unlikely.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0-1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [methyl-3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203-218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching addition). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching addition). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood: II. Mexiletine and flecainide.

A stabilized therapeutic drug monitoring procedure incorporating the novel Empore solid-phase extraction membrane (SPEM) for isolation of the antiarrhythmic drugs mexiletine (MEX) and flecainide (FLEC) from serum is described. Routinely, serum (0.5 ml), adjusted to pH 4.5, is passed through an octyl (C8) SPEM to extract the drugs. A methanol:water wash follows to remove proteins and interferences. MEX and FLEC are eluted from the membrane with mobile phase and an aliquot is injected directly onto a Zorbax cyanopropyl (CN) high-performance liquid chromatographic column with detection at 214 nm. Evaporating/concentrating techniques that can adversely influence the stability of the volatile MEX are unnecessary. Recovery for both drugs exceeds 90% and the assay is linear from 0.05 mg/L up to at least 6.0 mg/L for MEX and from 0.05 mg/L up to at least 3.0 mg/L for FLEC. Precision (between-run) coefficients of variation range from 2.3 to 3.0% (0.49–1.97 mg/L) for MEX and 3.7 to 5.9% (0.240–0.992 mg/L) for FLEC. Interferences are minimal. When we compared performance of the Empore SPEM and large-particle solid-phase sorbents packed in cartridges, we observed greater capacity per gram of sorbent and smaller elution volume with the membrane. Most important, concentrating steps that adversely affect the stability of MEX are avoided with the SPEM.