Improving LC/MS/MS-based bioanalytical method performance and sensitivity via a hybrid surface barrier to mitigate analyte - Metal surface interactions.

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification.

Use of Cyclic Ion Mobility Spectrometry (cIM)-Mass Spectrometry to Study the Intramolecular Transacylation of Diclofenac Acyl Glucuronide.

1-ß-O-Acyl-glucuronides (AGs) are common metabolites of carboxylic acid-containing xenobiotics, including, e.g., many nonsteroidal anti-inflammatory drugs (NSAIDs). They are of concern to regulatory authorities because of the association of these metabolites with the hepatotoxicity that has resulted in drug withdrawal. One factor in assessing the potential risk posed by AGs is the rate of transacylation of the biosynthetic 1-ß-O-acyl form to the 2-, 3-, and 4-O-acyl isomers. While transacylation can be measured using 1H NMR spectroscopy or liquid chromatography-mass spectrometry (LC-MS), the process can be time consuming and involve significant method development. The separation of these positional isomers by ion mobility spectrometry (IMS) has the potential to allow their rapid analysis, but conventional instruments lacked the resolving power to do this. Prediction of the collision cross section (CCS) using a machine learning model suggested that greater IMS resolution might be of use in this area. Cyclic IMS was evaluated for separating mixtures of isomeric AGs of diclofenac and was compared with a conventional ultraperformance liquid chromatography (UPLC)-MS method as a means for studying transacylation kinetics. The resolution of isomeric AGs was not seen using a conventional traveling wave IMS device; however, separation was seen after several passes around a cyclic IMS. The cyclic IMS enabled the degradation of the 1-ß-O-acyl-isomer to be analyzed much more rapidly than by LC-MS. The ability of cyclic IMS to monitor the rate of AG transacylation at different pH values, without the need for a prior chromatographic separation, should allow high-throughput, real-time, monitoring of these types of reactions.

Hybrid organic/inorganic hybrid surface technology for increasing the performance of LC/MS(MS)-based drug metabolite identification studies: Application to gefitinib and metabolites in mouse plasma and urine.

The detection, identification and quantification of drug metabolites plays a key role in drug discovery and development. Liquid chromatography (LC) coupled to mass spectrometry (MS) has become the primary technology for these studies due to its sensitivity and specificity. However, the presence of transition metals in the chromatography system and columns can result in non-specific and unwanted interactions with the drug and/or its metabolites, via electron-pair donation, leading to poor chromatography and analyte loss. The use of a hybrid organic/inorganic surface applied to the metal surfaces of the chromatography system and column has been demonstrated to reduce or eliminate these effects. When employed for the analysis of mouse urine, derived from the oral dosing of mice with the EGFR inhibitor gefitinib, we observed more symmetrical LC peaks. This resulted in a 33 % improvement in peak capacity for a 10 min reversed - phase gradient separation, a two-fold increase in MS response, cleaner MS spectra and improved peak response reproducibility. This hybrid surface barrier appears to offer significant advantages in the analysis of low-concentration metabolites, potentially facilitating the accurate determination of the elimination phase of the pharmacokinetic (PK) curve and detection of drug metabolites in microdosing or microsampling studies.

Rapid determination of the pharmacokinetics and metabolic fate of gefitinib in the mouse using a combination of UPLC/MS/MS, UPLC/QToF/MS, and ion mobility (IM)-enabled UPLC/QToF/MS.

The metabolism and pharmacokinetics of gefitinib (Iressa®, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholino-propoxy)quinazolin-4-amine), a selective thymidylate kinase inhibitor for the epidermal growth factor receptor (EGFR), was studied after IV and PO administration to male C57BL6 mice at 10 and 50 mg/kg respectively.The pharmacokinetics and metabolism of gefitinib were investigated using a range of rapid UHPLC-MS and UHPLC-IM-HRMS methods, using both reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC), to rapidly determine the drugs pharmacokinetics and metabolic fate.Rapid oral absorption resulted in peak plasma concentrations at 1 h of ca. 7 µg/mL, that declined with a half-life of 3.8 h (2.6 h for the IV route), and providing an estimated oral bioavailability of 53%. Gefitinib itself was the major circulating drug-related compound in plasma extracts, with a total of 11 metabolites identified.The urinary profiles determined using both HILIC and RP-UPLC-IM-MS detected gefitinib and 10 metabolites or 15 metabolites respectively including the detection of a number of novel glucuronide conjugates.Despite rapid, sub 5 min, LC profiling methods being employed metabolite coverage was shown to be high and compared well with that of previous studies.

Rapid profiling method for the analysis of lipids in human plasma using ion mobility enabled-reversed phase-ultra high performance liquid chromatography/mass spectrometry.

The incorporation of ion mobility (IM) into LC-MS analysis has been demonstrated to result in the generation of superior quality MS and MS/MS spectral data as well as providing enhanced resolution in the IM dimension based on lipid class. Here a sub 4 min microbore LC-ion mobility-accurate mass MS (LC-IM-MS) method has been developed for the rapid, profiling of lipids in biological fluids. The method was scaled directly from a conventional, 12  min, LC-MS analysis maintaining the chromatographic performance and lipid separation observed in the longer methodology giving a 75% saving in mobile phase consumption and analysis time. Because of the additional dimension of separation provided by IM, improvements in mass spectral quality from the increased resolution of co-eluting species were also seen when compared to the same separation without IM, thus aiding the identification of target lipids. When applied to human plasma samples some 5037 (positive ESI) and 2020 (negative ESI) mass/retention time features were detected following adduct deconvolution and, of these, 3727 and 800 of those present in the pooled plasma QC samples had a CV of below 30% for positive and negative ESI modes respectively. The method was applied to the analysis of a pilot set of commercially sourced breast cancer plasma samples enabling the differentiation of samples from healthy controls and patients based on their lipid phenotypes. Analysis of the resulting data showed that phosphatidylcholines, triglycerides and diglycerides exhibited lower expression and phosphatidylserine showed increased expression in the breast cancer samples compared to those of healthy subjects. The coefficients of variation, determined by reference to the QC data, for all of the features identified as potential markers of disease, were 6% or less.

Development of a rapid profiling method for the analysis of polar analytes in urine using HILIC-MS and ion mobility enabled HILIC-MS.

INTRODUCTION: As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC-MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. OBJECTIVES: To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. METHODS: A rapid untargeted HILIC LC-MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. RESULTS: The rapid HILIC-MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC-IMS-MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS information compared against the Metlin MS/MS database and, in addition, both calculated and measured CCS values from an experimentally derived CCS database. CONCLUSION: A rapid metabolic profiling method for the analysis of polar metabolites has been developed. The method has the advantages of speed and both reducing sample and solvent consumption compared to conventional profiling methods. The addition of IMS added an additional dimension for feature detection and the identification of metabolites.