Gary Lensmeyer | Of Wisconsin Hospital (original) (raw)

Papers by Gary Lensmeyer

Journal of Chromatography A, 1995

Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using t... more Cortisol, cortisone, corticosterone, prednisone and prednisolone are extracted from serum using the novel particle-loaded octyl (Cs)-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 (CIs) HPLC column. Method performance data demonstrate linearity from 0.4/~g/dl (low limit of detection) up to at least 60/xg/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. * Corresponding author. 0(121-9673/95/$09.50

Therapeutic Drug Monitoring, 2005

The authors describe an isocratic liquid chromatographic/electrospray single quadrupole mass spec... more The authors describe an isocratic liquid chromatographic/electrospray single quadrupole mass spectrometric assay suitable for routine therapeutic monitoring of the antirejection drugs tacrolimus, sirolimus, and cyclosporine in blood. The drugs and added internal standards, ascomycin, desmethoxysirolimus, and cyclosporine G, are extracted from a protein-free supernatant of patient blood on a Strata SDB-L styrene-divinylbenzene polymeric extraction cartridge (Phenomenex, Torrance, CA). A Gilson Aspec XL-4 (Gilson Instruments, Middleton, WI) is programmed to perform the extraction and transfer the extract to autosampler vials. The extract is automatically injected onto a C18 column held at 75 degrees C. Mobile phase, acetonitrile/water 90/10 (vol/vol), is pumped at 0.5 mL/min through a silica saturating column positioned in the column oven before the injector valve. Detection is by selected ion monitoring of the sodium adduct of each analyte, [M + 23]. A linear response is achieved for tacrolimus and sirolimus from limit of quantitation (LOQ) 1.0 microg/L to at least 80.0 microg/L. Cyclosporine was linear from LOQ 25 microg/L to at least 2000 microg/L. Interferences and ionization suppression are minimal. Analytic recovery for cyclosporine is 99.9 +/- 6.2% over a range of 104-1162 microg/L; sirolimus 107.7 +/- 9.3% over a range of 3.2-29.2 microg/L; and tacrolimus 101.9 +/- 2.5% over a range of 2.9-38.1 microg/L. Between-run relative standard deviations are 3.0% to 5.1% over a range of 9.8 microg/L to 30.7 microg/L for tacrolimus; 5.5% to 7.6% over a range of 13.5 microg/L to 35.2 microg/L for sirolimus; and 2.6% to 4.3% over a range of 186.0 microg/L to 1428.7 microg/L for cyclosporine. In the authors&amp;amp;amp;#39; laboratory, the method is robust and shows improved selectivity, precision, detection limits, and lower cost per test over available immunoassays. The authors find the method suitable for routine monitoring and therapy management of these drugs with over 50,000 samples analyzed over the course of 1 year.

Transplantation, 1986

There is much controversy as to whether the analysis of cyclosporine (CsA) should be performed by... more There is much controversy as to whether the analysis of cyclosporine (CsA) should be performed by radioimmunoassay (RIA) or high-performance liquid chromatography (HPLC), and whether the specimen should be serum or whole blood. Whole-blood specimens present specific advantages, but the presence of hemoglobin (Hgb) and other endogenous compounds can produce major errors in the RIA by "quenching" the analytical signal or by interfering with the antigen-antibody binding in the assay. We have developed a simple pretreatment step to remove the Hgb and other proteins responsible for this error. Red cells in whole blood are hemolyzed with a mixture of acetonitrile and water, the protein precipitated with acetonitrile, and the supernatant assayed by RIA. In a controlled study in which CsA concentration was kept constant and the Hgb concentration varied, the errors in measurement were directly proportional (r = 0.999) to the Hgb concentration. CsA values were spuriously deflated or inflated by 22.7 micrograms/L for each gram per 100 milliliters that the Hgb deviated from the 9.2 g/100 ml Hgb in the CsA calibration standards. In a similar study in which patient samples (n = 57) were assayed with and without pretreatment, the fractional error induced by Hgb was compounded in some patients by additional interferences that also appear to be removed by sample pretreatment. Without the pretreatment, CsA values could be in error by 33% when the Hgb varied 4 g/100 ml, thus providing potentially misleading results to the clinician. An I-125-labeled CsA tracer (purported not to be affected by the "quenching" interference of Hgb) produced consistently higher results when it was substituted for the tritiated CsA tracer contained in the Sandoz kit. In summary, sample pretreatment appears to be the simplest method of effectively removing endogenous interferences and minimizing erroneous results from whole blood submitted to the Sandoz RIA for CsA analysis.

sensitivity comparable with other HPLC methods for methyixanthines and is suitable for both thera... more sensitivity comparable with other HPLC methods for methyixanthines and is suitable for both therapeutic drug monitoring and pharmacokinetics studies. Because the specificity of the assay, in the absence of any extraction procedure, relies on the chromatographic separation, we have given special care to examining the possibility of co-eluting peaks. Theophylline and caffeine are well resolved from dyphylline and doxofylline, and none of 76 drugs we examined has shown significant interferences.

Therapeutic Drug Monitoring, 1995

The anticonvulsant drug gabapentin and its heptaneacetic acid analog-used here as an internal sta... more 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 further 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 sorbens as companion extraction devices.

Therapeutic Drug Monitoring, 1992

We describe the use of a new form of solid-phase material, the Empore solid-phase extraction memb... more We describe the use of a new form of solid-phase material, the Empore solid-phase extraction membrane (SPEM), for therapeutic drug monitoring. We evaluated the new extraction procedure with the companion high-performance liquid chromatographic (HPLC) method for the antiarrhythmic drug amiodarone and its metabolite, desethylamiodarone, in patients' serum. Acidified serum (250 microliters) was passed through an octyl (C8) SPEM secured in an MF-1 microfilter unit. Serum proteins and potential interferences were removed with an acetonitrile:water wash, and the retained drugs eluted with HPLC mobile phase. This eluate was injected directly onto the analytical column. Both drugs averaged 85% recovery with a linear response from a lower limit of detection at 0.05 mg/L up to 6 mg/L, and between-run precision coefficients of variation ranging from 3.1 to 6.4% over the concentration range of 0.5-3.0 mg/L. We observed significant advantages of the novel SPEM over conventional liquid-liquid or large-particle size solid-phase sorbents packed in cartridges. Minimal amounts of solvents were required, elution volume was smaller, time-consuming evaporating/concentrating steps that can influence drug stability were avoided, and little throw-away material was generated. Only the small membrane was discarded.

Veterinary Surgery, 1998

Objective—To compare two methods of whole blood cyclosporine A (CsA) measurement in cats.Study De... more Objective—To compare two methods of whole blood cyclosporine A (CsA) measurement in cats.Study Design—Whole blood samples were analyzed for CsA concentrations with use of high performance liquid chromatography (HPLC) and monoclonal immunoassay methods.Animals—Blood (n = 36 samples) was obtained from six cats after renal transplantation.Methods—Results were compared by linear regression analysis using both pooled and individual patient data. Eight samples were off-scale on the immunoassay and were excluded.Results—There was significant correlation between CsA measured using HPLC and immunoassay methods (P < .001; r= .942; r2= .887). However, individuals varied nonrandomly from the mean pooled patient data. Correlation between the assay methods was higher for individual patients using data only from that specific individual (mean r value = .976; r2= .955). Clinical utility of the immunoassay (ie, results would prompt an appropriate CsA dosage adjustment) was good when based on individually derived conversion factors (27 of 28 [96.5%] of decision events).Conclusion—HPLC is superior for measurement of blood CsA concentrations in cats after kidney transplantation. However, an immunoassay may provide reliable information for CsA management if a comparative database (HPLC v immunoassay) has been previously determined in a specific patient.Clinical Relevance—Locally available monitoring of CsA by immunoassay in cats may provide significant advantages when shipping of blood samples to distant locations is required to obtain analysis by HPLC. These advantages may include cost and timeliness of results in circumstances where daily blood CsA concentrations may be desired, such as when managing an acute rejection reaction.

Epilepsy Research, 1997

Drug interactions can significantly complicate the management of patients receiving multiple medi... more 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.

Chemicals. Acetonitrile was "HPLC" grade from J. T. Baker Chemical Co., Phillipsburg, NJ. Distill... more Chemicals. Acetonitrile was "HPLC" grade from J. T. Baker Chemical Co., Phillipsburg, NJ. Distilled de-ionized water was prepared with the "Milli Q"water purification system (Millipore Corp., Bedford, MA). Solutions of water! Drug-free whole-blood samples supplemented with the cyclosporines and samples from 10 transplant patients receiving cyclosporin A (C5A) were equilibrated at 4, 22, and 37 #{176}C for 2.5 h; the plasma and cells were separated; and the fractions were assayed by high-performance liquid chromatography (HPLC). Partitioning of GsA and metabolites among plasma and cells was diverse and not always predictable for patients' samples. Overall, although widely variable, >50% of the total concentration of metabolites Ml, M8, Mg, Ml 0, M16, M17, Ui, U8, and U9 in whole blood was associated with the cells, whereas >50% of M13, M18, M21, M25, M26, M203-218, U2, U3, U4, U5, U6, and U7 was associated with plasma. A decrease in hematocrit from 47.8% to 24%, an increase of the sample's temperature (from 4 to 37 #{176}C), or an increase in analyte concentration (usually >500 g/L for selected metabolites) increased the relative portion associated with plasma in a nonlinear fashion. Parent CsA was most influenced by these changes; its relative concentrations in plasma varied from 18% to 50%. These data support the preferential use of whole blood for therapeutic monitoring of "cyclosporines."Through additional studies, we suggest possible mechanisms affecting the distribution phenomenon and ascribe chemical structure-distribution relationships. AddItional Keyphrases: variation, source of sample handling organ transplants . immunosuppressive drugs . chromatography, liquid . metabolism

We demonstrate the diverse selectivity of three commercial polyclonal "cyclosporine" immunoassays... more We demonstrate the diverse selectivity of three commercial polyclonal "cyclosporine" immunoassays for cyclosporin (CsA) metabolites by comparing analytical responses of nine metabolites added to drug-free whole-blood specimens (range 0 to 2000 pg/L) and assayed by the Abbott TDx fluorescence polarization immunoassay (FPIA), the Incstar Cyclo-Trac radioimmunoassay (AlA), and the Sandoz AlA. Cross-reactivity--defined as the relative response (slope of regression line) of metabolite/parent CsA over the assay's linear range of concentrations-differed for each metabolite among the three assays. Overall, Abbott's antiserum exhibited the greatest affinity for the metabolites, the Sandoz antiserum the least. Ranges of cross-reactivity for the metabolites over all three assays were Ml (14-44%), M8 (9-20%), M13 (13-26%), M17 (50-116%), M18 (17-79%), M21 (4-54%), M25 (<1-52%), M26 (<1-29%), and M203-218 (7-51%). The specificities of the Abbott, Incstar, and Sandoz polyclonal assays thus differ significantly, and this brings into question the practical utility of comparing data generated for patients' specimens by different procedures. Commercial immunoassays constitute a significant portion of all analytical methods for clinical measurement of "cyclosporine" (CsA). The performance of these assays varies, as evidenced by the poor precision (CV 21.9%) in a national interlaboratory CsA assessment program (1). Currently, CsA can be quantified by high-performance liquid chromatographic (HPLC), RIA, or fluorescence polarization immunoassay (F'PIA) procedures. Most HPLC methods are calibrated to measure only CsA itself. Assays for select metabolites have been described but are not yet considered more clinically useful. In polyclonal BIAs from Incstar (CYCLO-Trac) and Sandoz, im1 and 3H-labeled CsA tracers, respectively, are used to detect and quantifr antigen-antibody interactions.

Journal of Pharmaceutical Sciences, 1985

Journal of Pharmaceutical Sciences, 1984

Volume 5 of this series is devoted entirely to a review of sesquiterpene total synthesis from 197... more Volume 5 of this series is devoted entirely to a review of sesquiterpene total synthesis from 1971 to 1979. Those familiar with the literature on synthesis will appreciate the enormity of this task. In Volume 2 a single chapter covered sesquiterpene synthesis through 1970; that an entire volume is now required attests to the expIosion of research in this area during the past decade. The Heathcock group has assembled a lucid and readable account of modem sesquiterpene synthesis which can be heartily recommended to students and practicing chemists alike.

Veterinary Surgery, 1999

Objective-To determine the effects of ketoconazole (KC) on the pharmacokinetics of cyclosporine A... more Objective-To determine the effects of ketoconazole (KC) on the pharmacokinetics of cyclosporine A (CsA) elimination in cats. Study Design-Research study and prospective clinical trial. Animals-Five healthy adult cats (pharmacokinetic studies) and 6 client-owned cats with chronic renal failure. Methods-Blood CsA concentrations were measured after CsA (4 mg/kg IV) administration with or without concurrent oral KC (10 mg/kg). Subsequently, a combined CsA-KC immunosuppressive regimen was used in cats after kidney transplantation. Blood CsA concentrations were measured using high performance liquid chromatography. CsA elimination was analyzed using a computerized pharmacokinetics program. Results-KC increased blood CsA concentrations 1.8-fold and 2.2-fold at 12 and 24 hours after CsA administration. KC significantly decreased the mean systemic CsA clearance from 2.73 mL/min/kg to 1.22 mL/min/kg resulting in an increase in the terminal phase half-life from 10.7 to 22.2 hours. The volume of distribution of steady-state of CsA was unaffected by KC. In a series of clinical feline kidney transplant patients, a once-a-day CsA-KC regimen was able to be used in most of the cats and was effective for prevention of allograft rejection in all of these cats. Conclusion and Clinical Relevance-KC is an effective adjunct treatment for immunosuppression in feline kidney transplant patients. KC suppresses CsA elimination, which reduces the need for CsA and allows once daily administration of CsA.

Epilepsy Research, 1996

The anticonvulsant gabapentin is transported across biological membranes via the L-amino acid tra... more The anticonvulsant gabapentin is transported across biological membranes via the L-amino acid transport system (System-L). Absorption of gabapentin is saturable, and in-vitro data have previously demonstrated that both L-leucine and L-phenylalanine may compete with the intestinal transport of gabapentin. The purpose of this study therefore was to determine whether a high-protein meal would interfere with gabapentin absorption. Ten healthy volunteers received in a randomized, cross-over design, a single 600-mg dose of gabapentin in the fasting state and after a high-protein meal consisting of 80 gm total protein (4.1 g phenylalanine, 8.2 g leucine and 4.2 g isoleucine), 52 g carbohydrate, and 9 g fat. Plasma gabapentin concentrations were measured by HPLC at baseline, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 30 h. Calculated pharmacokinetic parameters included Cma x, Tma x, AUC and Tl/2. In addition, a pharmacodynamic assessment (using visual analog scales) of gabapentin-related adverse effects was performed at 2 h post drug ingestion and was compared between study phases. Statistical analysis included Student's t-test for paired data, with significance assigned at P < 0.05. Cma ~ was significantly increased by 36% (3.87 -+ 1.15 vs 5.28 + .97/xg/ml, P = 0.002), and Tin, x tended to be shorter (3.9 + 1.8 vs 2.8 + .35 h, P = 0.10), after the high-protein meal. Although AUC was increased by 1 I%, this did not achieve statistical significance. Despite significantly higher plasma concentrations at 2 h, subjects reported significantly fewer adverse effects after the high-protein meal.

A rapid, simple, accurate, and precise isothermal gas-chromatographicmethod is introducedfor dete... more A rapid, simple, accurate, and precise isothermal gas-chromatographicmethod is introducedfor determination of methaqualone (2-methyl-3-o-tolyl-

Clinical Toxicology, 1977

Clinical Chemistry, 2006

Results: For the new HPLC assay, between-run CVs were 2.6%-4.9% for 25(OH)D 3 and 3.2%-13% for 25... more Results: For the new HPLC assay, between-run CVs were 2.6%-4.9% for 25(OH)D 3 and 3.2%-13% for 25(OH)D 2 ; recoveries were 95%-102%; and the assay was linear from 5 g/L to at least 200 g/L. Comparison data were as follows: for HPLC vs LC-MS/MS, y ‫؍‬ 1.01x ؊ 4.82 g/L (S yͦx ‫؍‬ 4.93 g/L; r ‫؍‬ 0.996) for 25(OH)D 3 , and y ‫؍‬ 0.902x ؊ 0.566 g/L (S yͦx ‫؍‬ 2.56 g/L; r ‫؍‬ 0.9965 for 25(OH)D 2 ; for HPLC vs Diasorin RIA, y ‫؍‬ 0.709x ؊ 5.86 g/L (S yͦx ‫؍‬ 7.35 g/L; r ‫؍‬ 0.7509); and for HPLC vs Nichols Advantage CPBA, y ‫؍‬ 1.00x ؊ 3.60 g/L (S yͦx ‫؍‬ 32.7 g/L; r ‫؍‬ 0.6823).

Therapeutic Drug Monitoring, 1991