Matthew S. Halquist

Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations.

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.

Quantification of Alefacept, an immunosuppressive fusion protein in human plasma using a protein analogue internal standard, trypsin cleaved signature peptides and liquid chromatography tandem mass spectrometry.

Quantitative analysis of a therapeutic protein through use of surrogate proteotypic peptides was evaluated for the measurement of Amevive (Alefacept) in human plasma using liquid chromatography tandem mass spectrometry. Signature peptides were obtained through in silico and iterative tuning processes to represent Alefacept for quantification. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with pH controlled at 5.1 and heat denaturation at 45°C followed by enzymatic digestion, dilution, and filtration. On-line extraction of the signature peptides was carried out using a Phenomenex Gemini C18 security guard column (4.0 mm × 2.0 mm) as a loading column and a Gemini C18 (100 mm × 2.1 I.D., particle size 5 μm) as the analytical (eluting) column. Tandem mass spectrometric detection was performed on a hybrid triple quadrupole linear ion trap equipped with electrospray ionization to positively ionize signature peptides for Alefacept and myoglobin. The method was linear for Alefacept (protein) concentrations between 250 and 10,000 ng/mL. Precision and accuracy for inter- and intra-assay for the lower limit of quantification was less than 20% (16.2 and 10.3, respectively). The method was validated according to current FDA guidelines for bioanalytical method validation.

A modified method for the determination of tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine by solid phase extraction using a molecularly imprinted polymer and liquid chromatography tandem mass spectrometry

The work described in this paper represents an improvement over a previously published method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine by solid phase extraction on a molecularly imprinted polymer column coupled with HPLC and -MS/MS detection. The influence of ion suppression due to sample matrix effect was evaluated, and found to influence the response of NNAL. By changing the liquid chromatography conditions, the response for this method was enhanced approximately 25-fold through avoidance of ionization suppression that was found with a previously published method and sample throughput has been improved. The dynamic range of the assay extends from 20 to 2500 pg/mL with a mean r(2)>0.998. The lower limit of quantitation for the assay was 20 pg/mL despite the use of an inherently lower sensitivity instrument. The method was validated according to current FDA guidelines for bioanalytical method validations.

Quantitative liquid chromatography tandem mass spectrometry analysis of macromolecules using signature peptides in biological fluids

Targeted protein quantification using peptide surrogates has increasingly become important to the validation of biomarker candidates and development of protein therapeutics. These approaches have been proposed and employed as alternatives to immunoassays in biological fluids. Technological advances over the last 20 years in biochemistry and mass spectrometry have prompted the use of peptides as surrogates to quantify enzyme digested proteins using triple quadrupole mass spectrometers. Multiple sample preparation processes are often incorporated to achieve quantification of target proteins using these signature peptides. This review article focuses on these processes or hyphenated techniques for quantification of proteins with peptide surrogates. The most recent advances and strategies involved with hyphenated techniques are discussed.

Determination of oxyntomodulin, an anorectic polypeptide, in rat plasma using 2D-LC-MS/MS coupled with ion pair chromatography.

Polypeptide therapeutics present a challenge for quantitative analysis when using immunoassays or recently, liquid chromatography-tandem mass spectrometry because of their structural similarities to endogenous proteins and peptides in plasma. In this assay, a Waters Oasis® mixed-mode anion exchange (MAX) microelution modified solid phase extraction (SPE) method coupled with two-dimensional reversed phase ion pair chromatography-tandem mass spectrometry was used for the validation and analysis of oxyntomodulin in rat plasma. Oxyntomodulin (OXM) and its isotope labeled internal standard were extracted from rat plasma and analyzed with a chromatographic run time of 8 min. Modified SPE, two-dimensional liquid chromatography coupled with 3-nitrobenzyl alcohol as a mobile phase additive, and monitoring of multiply charged SRM transitions (+7 charge state) of OXM were necessary to achieve a lower limit of quantification of 1 ng/mL. The method was validated with a linear range of 1-1000 ng/mL, with average R² of 0.992, and reversed calculated residuals between -8.6% and 6.0%. Precision and accuracy for inter- and intra-day were determined to be ±17%. Following a complete validation, the method was applied to show utility using rat plasma samples that were intravenously dosed with oxyntomodulin.

Assessment of matrix effects and determination of niacin in human plasma using liquid–liquid extraction and liquid chromatography–tandem mass spectrometry

A simple, sensitive and rapid liquid-liquid extraction method for the analysis of nicotinic acid (niacin) and its labeled internal standard nicotinic acid-d4 (niacin-d4) in human plasma was developed and validated. The analyte and its internal standard were isolated from acidified plasma using a single liquid-liquid extraction procedure with methyl-t-butyl ether. The extracted samples were analyzed by liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The calibration curves were linear in the measured range between 5 and 1000 ng/mL and the limit of detection was calculated as 122 pg/mL. The method required 250 microL of human plasma and the total run time between injections was 3.5 min. Matrix effects were assessed by post-column infusion experiments, phospholipids monitoring and post-extraction addition experiments. The extraction of phospholipids and niacin from plasma was studied under acidic, neutral and basic conditions. Acidic conditions were optimal for both the recovery of niacin and the removal of phospholipids; the degree of matrix effects for niacin was determined to be 2.5%. It was concluded that effective removal of matrix components can overcome low recovery issues associated with liquid-liquid extractions of polar analytes.

Simultaneous determination of gallic acid and gentisic acid in organic anion transporter expressing cells by liquid chromatography-tandem mass spectrometry.

In order to elucidate the role of organic anion transporters (OATs) in the renal elimination of gallic acid and gentisic acid, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of gallic acid and gentisic acid in cell lysate, using Danshensu as the internal standard (IS). After a simple liquid-liquid extraction, the analytes were detected in negative ESI mode using selected reaction monitoring. The precursor-to-product ion transitions (m/z) were 169.0→125.0, 153.1→108.0, and 196.8→135.2 for gallic acid, gentisic acid, and the IS, respectively. Chromatographic separation was achieved on a C18 column using mobile phases consisting of water with 0.1% acetic acid (A) and acetonitrile with 0.05% formic acid. (B) The total run time was 3min and calibration curves were linear over the concentrations of 0.33-2400ng/mL for both compounds (r(2)>0.995). Good precision (between 3.11% and 14.1% RSD) and accuracy (between -12.7% and 11% bias) was observed for quality controls at concentrations of 0.33 (lower limit of quantification), 1, 50, and 2000ng/mL. The mean extraction recovery of gallic acid and gentisic acid was 80.7% and 83.5%, respectively. Results from post-column infusion and post-extraction methods indicated that the analytical method exhibited negligible matrix effects. Finally, this validated assay was successfully applied in a cellular uptake study to determine the intracellular concentrations of gallic acid and gentisic acid in OAT expressing cells.

Safety studies of post-surgical buprenorphine therapy for mice

The use of appropriate analgesia in laboratory mice may be suboptimal because of concerns about adverse events (AE). Target Animal Safety trials were conducted to determine the safety of an extended-release suspension of buprenorphine. Drug or control suspensions were injected subcutaneously in surgically-treated BALB/c mice anesthetized with ketamine–xylazine to mimic post-operative conditions in which the compound might commonly be administered. Single and repeat five-fold (5×) excesses of the 3.25 mg/kg intended dose were used to provoke potential AE. Trials included prospective measurements of weight changes, blood chemistry, hematology, and histopathology. Clinical and histopathology findings were similar in drug-treated and control mice in a four-day trial using a single 16.25 mg/kg, 5× overdose of the drug. In a 12-day trial, which used a total buprenorphine dose of 48.75 mg/kg, clinical and histopathology values were also similar in control and drug-treated female mice. In the male arm of the repeat-overdose trial, two of eight mice died on the morning of day 12, three days following the third 16.25 mg/kg overdose administration. Histopathology did not reveal a cause of death. In a 14-month trial using a single 3.25 mg/kg dose of the drug, no significant findings identified potential AE. These findings indicate a high tolerance to an extended-release buprenorphine suspension administered post-operatively in mice with appropriate husbandry.

A Simple High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Monoamine Neurotransmitters and Relative Metabolites with Application in Mouse Brain Tissue

As a means to evaluate the role of organic solute carriers in CNS homeostasis, a simple and rapid high-performance liquid chromatographic method utilizing ultraviolet and electrochemical detectors was developed and validated for the simultaneous determination of the neurotransmitters dopamine, norepinephrine, and serotonin and their metabolites in mouse brain homogenate. For analyte separation, the method utilized a C18 column with a mobile phase of 75 mM sodium dihydrogen phosphate (monohydrate), 1.7 mM 1-octanesulfonic acid sodium salt, 25 µM EDTA, 10% acetonitrile, and 1% triethylamine with a final pH of 3.0 adjusted using phosphoric acid. The method was linear for dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from 20–1000 ng/mL (r ≥ 0.993) with detection limits of 5 ng/mL for dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, and of 20 ng/mL for serotonin and 5-hydroxyindoleacetic acid. The method was linear over the range of 10–200 µg/ml for quinolinic acid, xanthurenic acid, and nicotinic acid (r ≥ 0.993) with detection limits of 1 µg/mL for all components. At a flow rate of 1 mL/min, the analytical run time was less than 10 min. The method was successfully applied to the quantitation of these compounds in mouse whole brain homogenates.

Quantitative determination of buprenorphine, naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0-1000 ng/mL. The intra and inter-day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra- and inter-day accuracy (bias) ranged from -14.2 to 15.2% and from -9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC-MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine-naloxone combination in rats.