Allena Ji

A sensitive human bone assay for quantitation of tigecycline using LC/MS/MS

Tigecycline (Tygacil,Wyeth) is a first-in-class, broad spectrum antibiotic with activity against multiple-resistant organisms. In order to address the unexpectedly low tigecycline concentrations in human bone samples analyzed using a LC/MS/MS method developed elsewhere, we have developed and validated a new and sensitive human bone assay for the quantitation of tigecycline using LC/MS/MS. The new method utilizes the addition of a stabilizing agent to the human bone sample, homogenization of human bone in a strong acidic-methanol extraction solvent, centrifugation of the bone suspension, separation by liquid chromatography, and detection of tigecycline by mass spectrometry. Linearity was demonstrated over the concentration range from 50 to 20,000 ng/g using a 0.1g human bone sample. The intra- and inter-day accuracy of the assay was within 100+/-15%, and the corresponding precision (CV) was <15%. The stability of tigecycline was evaluated and shown to be acceptable under the assay conditions. The extraction recovery of tigecycline with the current method was 79% when using radio-labeled rat bone samples as a substitute for human bone samples. Twenty-four human bone samples collected previously from volunteers without infections who had elective orthopedic surgery after receiving a single dose of tigecycline were re-analyzed using the current validated method. Tigecycline concentrations in these samples ranged from 238 to 794 ng/g with a mean value 9 times higher than the mean concentration previously reported. The data demonstrated that the current method has significantly higher extraction efficiency than the previously reported method.

Determination of tigecycline in human skin using a novel validated LC–MS/MS method

BACKGROUND To develop and validate a sensitive and novel bioanalytical method for measuring tigecycline concentrations in human skin using LC-MS/MS. RESULTS The method utilizes addition of a stabilizing agent to the human skin or surrogate (human liver or rat skin), homogenization of human skin in a strong acidic-methanol extraction solvent, centrifugation of the skin suspension, filtration of the skin suspension supernatant, separation by LC (Polaris™ C18-A 50 × 2.0 mm), and detection of tigecycline by MS/MS. Linearity was 50-20,000 ng/g, using a sample size of 100 mg. The intra-and inter-day accuracy and precision of the assay met acceptance criteria. CONCLUSION This method has been successfully applied to 17 incurred human skin samples from volunteers with surgical infections who received intravenous doses of tigecycline (100 mg initial loading dose and 50 mg every 12 h for at least 2 days). Tigecycline concentrations in these samples ranged from 185 to 2853 ng/g.

Reassessment of tigecycline bone concentrations in volunteers undergoing elective orthopedic procedures.

The goal of the this study was to re‐evaluate tigecycline bone concentrations in subjects undergoing elective orthopedic surgery, using multiple doses and a more robust bone assay than was used in a previous study. Each subject received three intravenous doses of tigecycline (one 100‐mg infusion followed by two 50‐mg infusions, each administered over 30 minutes). A single bone sample was collected from each subject at one of the following times: 1, 2, 4, 6, 8, or 12 hours after the third dose. Four blood samples were collected from each subject: before the first dose, before and after the third dose, and within 15 minutes of the collection time of the bone sample. Noncompartmental pharmacokinetic analysis serum and bone area under the curve for the given dose interval (AUCτ) values were 2,402 ng h/mL and 11,465 ng h/g, and maximum concentration (Cmax) values were 974 ng/mL and 2,262 ng/g, respectively. The bone to serum ratio calculated using the AUCτ values was 4.77, confirming tigecycline penetration into bone.

A novel approach for quantitation of glucosylceramide in human dried blood spot using LC-MS/MS.

BACKGROUND Glucosylceramide, an efficacy biomarker for Gaucher Type 1 disease, exhibits poor solubility in polar solvents and whole blood which makes it difficult to prepare a homogenous blood standard. RESULTS We developed a novel method using standard addition approach by spiking a small volume of analyte solution on the surface of prespotted dried blood spot. The whole spots were punched out for subsequent extraction and LC-MS/MS analysis. The assay performance met all validation acceptance criteria. Glucosylceramide concentrations in 50 paired plasma and dry blood spot samples obtained from Gaucher Type 1 patients were tested and the results demonstrated the feasibility of using the DBS method for clinical biomarker monitoring. CONCLUSION The new approach greatly improves assay precision and accuracy.

Ultrasensitive and automated 1 pg/ml fluticasone propionate assay in human plasma using LC–MS/MS

BACKGROUND To develop and validate an ultrasensitive bioanalytical assay for quantitation of fluticasone propionate in human plasma, aliquots of 0.6 ml of K(3)EDTA human plasma were treated with zinc sulfate solution and loaded onto a preconditioned SPE plate. The sample solutions were washed, eluted, dried and reconstituted. The extracted sample was injected onto a LC-MS/MS system and separated by a reverse-phase HPLC column with a 5 min gradient program, and detected by MS/MS for fluticasone propionate quantitation. RESULTS Linearity was from 1 to 200 pg/ml. The intra- and inter-day accuracy and precision of the assay met validation acceptance criteria. Various stabilities were established and interference drug assessment was evaluated. The assay has been used for clinical studies. CONCLUSION This ultrasensitive method has been successfully validated using LC-MS/MS for determination of fluticasone propionate in human plasma at low pg/ml level.

Enhancement of human plasma glucosylceramide assay sensitivity using delipidized plasma.

Glucosylceramide (GL-1) level in human has been considered as a surrogate biomarker for enzyme replacement and substrate reduction therapies (ERT and SRT) for Gaucher and Fabry patients. Due to the high endogenous level of GL-1 in human plasma, it is difficult to achieve the analytical sensitivity of plasma GL-1 below the normal endogenous level (1.7 μg/mL to 6.6 μg/mL) when using the standard addition method and regular plasma matrix for standard curve. A high sensitivity plasma GL-1 assay with LLOQ at 0.1 μg/mL was developed and validated using delipidized plasma so that patient plasma concentrations that are below normal reference range can be measured accurately. The normal reference range was established from 120 healthy donors using this developed new method. Twenty-three Fabry patient plasma samples including baseline and post-investigation drug treatment samples were measured. All post-treatment samples showed GL-1 concentration below 2.0 μg/mL, indicating the utility of the reported high sensitivity assay using delipidized plasma for monitoring the plasma GL-1 biomarker level in patients.