Validation of reliable and selective methods for direct determination of glyphosate and aminomethylphosphonic acid in milk and urine using LC-MS/MS.

Simple high-throughput procedures were developed for the direct analysis of glyphosate [N-(phosphonomethyl)glycine] and aminomethylphosphonic acid (AMPA) in human and bovine milk and human urine matrices. Samples were extracted with an acidified aqueous solution on a high-speed shaker. Stable isotope labeled internal standards were added with the extraction solvent to ensure accurate tracking and quantitation. An additional cleanup procedure using partitioning with methylene chloride was required for milk matrices to minimize the presence of matrix components that can impact the longevity of the analytical column. Both analytes were analyzed directly, without derivatization, by liquid chromatography tandem mass spectrometry using two separate precursor-to-product transitions that ensure and confirm the accuracy of the measured results. Method performance was evaluated during validation through a series of assessments that included linearity, accuracy, precision, selectivity, ionization effects and carryover. Limits of quantitation (LOQ) were determined to be 0.1 and 10 µg/L (ppb) for urine and milk, respectively, for both glyphosate and AMPA. Mean recoveries for all matrices were within 89-107% at three separate fortification levels including the LOQ. Precision for replicates was ≤ 7.4% relative standard deviation (RSD) for milk and ≤ 11.4% RSD for urine across all fortification levels. All human and bovine milk samples used for selectivity and ionization effects assessments were free of any detectable levels of glyphosate and AMPA. Some of the human urine samples contained trace levels of glyphosate and AMPA, which were background subtracted for accuracy assessments. Ionization effects testing showed no significant biases from the matrix. A successful independent external validation was conducted using the more complicated milk matrices to demonstrate method transferability.

Identifying and overcoming bioanalytical challenges associated with chlorine-containing dehydrogenation metabolites.

Liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is a widely utilized analytical tool for quantifying small molecules in complex biological matrices. In certain situations the mass-selection capabilities of the tandem mass spectrometer may be insufficient to discriminate between the analyte of interest and its metabolites, particularly those metabolites that are isobaric with the analyte. One scenario by which isobaric interference may occur is the metabolism of a chlorine- or bromine-containing small molecule to a metabolite with the concomitant loss of 2 Da. This report describes the detection and characterization of two distinct dehydrogenation [M-2] metabolites during LC/MS/MS quantification of a chlorinated small molecule in rat plasma samples derived from a toxicokinetic study. The potential isotope-related impact of these metabolites on quantification of the parent compound was assessed. Several alternate precursor ion and product ion combinations were evaluated and shown to minimize the quantitative impact of the interfering metabolites without having to rely on their stringent chromatographic resolution from the parent compound. These results indicate that when quantifying chlorine- or bromine-containing small molecules from in vivo samples or in vitro metabolic incubations: (1) efforts to detect potential dehydrogenation metabolites should be undertaken and (2) if such metabolites are detected, the judicious choice of alternate multiple-reaction monitoring (MRM) transitions can limit their impact on quantification of the parent molecule without the need for robust chromatographic resolution.

Application of hydrophilic interaction chromatography retention coefficients for predicting peptide elution with TFA and methanesulfonic acid ion-pairing reagents.

Hydrophilic retention coefficients for 17 peptides were calculated based on retention coefficients previously published for TSKgel silica-60 and were compared with the experimental elution profile on a Waters Atlantis HILIC silica column using TFA and methanesulfonic acid (MSA) as ion-pairing reagents. Relative peptide retention could be accurately determined with both counter-ions. Peptide retention and chromatographic behavior were influenced by the percent acid modifier used with increases in both retention and peak symmetry observed at increasing modifier concentrations. The enhancement of net peptide polarity through MSA pairing shifted retention out by nearly five-fold for the earliest eluting peptide, compared with TFA. Despite improvements in retention and efficiency (N(eff)) for MSA over TFA, a consistent reduction in calculated selectivity (alpha) was observed. This result is believed to be attributed to the stronger polar contribution of MSA masking and diminishing the underlying influence of the amino acid residues of each associated peptide. Finally, post-column infusion of propionic acid and acetic acid was evaluated for their potential to recover signal intensity for TFA and MSA counter-ions for LC-ESI-MS applications. Acetic acid generally yielded more substantial signal improvements over propionic acid on the TFA system while minimal benefits and some further reductions were noted with MSA.

Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC-MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum.

PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC-MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k'), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of logk' versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC-MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.

Rapid determination of the applicability of hydrophilic interaction chromatography utilizing ACD labs log D suite: a bioanalytical application.

Hydrophilic interaction chromatography (HILIC) is an effective technique for retaining and separating polar compounds. This approach offers several advantages for bioanalytical liquid chromatography/mass spectrometry, considering that a majority of active pharmaceutical ingredients are polar amines. HILIC employs high concentrations of relatively polar organic mobile phase components (i.e. acetonitrile), providing enhanced desolvation and electrospray ionization efficiency, as well as allowing direct injection of many protein precipitation, liquid/liquid, and solid phase extracts. A set of 30 probe compounds was evaluated to demonstrate a relationship between a compound's HILIC capacity factor (k'), and pH dependent distribution coefficient (D), using three sets of generic isocratic conditions. Plots of logk' versus logD(pH3.0) produced correlation coefficients of 0.751, 0.696, and 0.689 at acetonitrile mobile phase concentrations of 85%, 90%, and 95% (v/v), respectively. For bioanalytical applications a k'>2 is typically targeted to ensure adequate retention of a given analyte relative to extracted matrix components. Using k'> or =2 as a measure of HILIC applicability, the linear relationships for each of the three acetonitrile levels predicted whether or not HILIC was able to meet this criterion for at least 90% of the compounds tested. Overall, the relationship between k' and logD can serve as a valuable tool for identifying the applicability of HILIC and a starting point for the chromatographic conditions, prior to the initiation of any laboratory activities. Additionally, this relationship can assist with the selection of appropriate chemical analog internal standards.

Novel Two-Stage Fine Milling Enables High-Throughput Determination of Glyphosate Residues in Raw Agricultural Commodities.

Dramatic process-efficiency gains for residue analysis of glyphosate in raw agricultural commodities (RACs) were achieved by development and validation of a two-stage fine-milling process. This secondary milling produced a uniform and consistent product that could be reproducibly measured with 75 mg analytical test portions. The milligram scale sample size enabled the direct weighing of sample into a liquid-handler-compatible 96-well format. A high-throughput workflow based on this innovative comminution approach for the quantitation of glyphosate, a nonselective herbicide, and its main degradation product, aminomethylphosphonic acid, was validated in various RACs and used to demonstrate the applicability of the two-step milling process. The precision and reproducibility of 75 mg analytical portions taken through this assay was used to demonstrate the feasibility of using a two-stage fine-milling technique for pesticide residue applications. An RSD of less than 10% was achieved in endogenous glyphosate residues in multiple RACs. Comparable recoveries and superior precision were achieved with this new method as compared with a validated 10 g scale method.

Quantitative analysis of dicamba residues in raw agricultural commodities with the use of ion-pairing reagents in LC-ESI-MS/MS.

A sensitive and selective HPLC-MS/MS method was developed for the quantitative analysis of dicamba residues in raw agricultural commodities (RACs). Instead of analysis in the traditionally used negative electrospray ionization (ESI) mode, these anionic compounds were detected in positive ESI with the use of ion-pairing reagents. In this approach, only a small amount (60µM) of a commercially available dicationic ion-pairing reagent was introduced into the post-column sample stream. This method has been validated in six different types of RACs including corn grain, corn stover, cotton seed, soybean, soy forage and orange with satisfactory quantitative accuracy and precision. The limits of quantitation (LOQ) values for these analytes were 1.0 to 3.0µg/kg. The standard curves were linear over the range of the tested concentrations (3.0 to 500µg/kg), with correlation coefficient (r) values≥0.999. Evaluation of ionization effects in RAC matrix extracts using diluent blanks for comparison showed no significant matrix effects were present.

Direct and sensitive determination of glyphosate and aminomethylphosphonic acid in environmental water samples by high performance liquid chromatography coupled to electrospray tandem mass spectrometry.

A novel method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the sensitive determination of glyphosate and its major degradation product, AMPA in environmental water samples. The method involves the use of MS compatible mobile phases (0.1% formic acid in water and acetonitrile) for HPLC and direct analysis of water samples without sample derivatization. The method has been validated in different types of water matrices (drinking, surface and groundwater) by accuracy and precision studies with samples spiked at 0.1, 7.5 and 90 ppb. All mean accuracy values ranged from 85% to 112% for glyphosate and AMPA using both primary and secondary quantitative ion transitions (RSD ≤ 10%). Moreover, both primary and secondary ion transitions for glyphosate and AMPA can achieve the quantitation limits at 0.1 ppb. The linear dynamic range of the calibration curves were from 0.1 to 100 ppb for each analyte at each ion transitions with correlation coefficient higher than 0.997.

Interlaboratory assessment of cryomilling sample preparation for residue analysis.

The effectiveness of the comminution approach used for bulk field samples limits the size of the subsample that must be extracted and analyzed to ensure an adequately representative and reproducible measurement. In many cases this subsample size restricts the residue method to the use of larger vessel formats, limiting downstream throughput. The introduction of a secondary fine-milling step to this process using a subsample size already known to be representative can further improve sample homogeneity and allow direct method scaling to small high-throughput formats. Dramatic increases in method throughput can then be achieved through the simultaneous processing of numerous samples in parallel. This approach was evaluated across a diverse grouping of crop matrices using two substantially different pesticide types. Both fortified and field-collected samples demonstrated a high degree of precision and reproducibility across laboratories. Additional benefits of this approach include significant reductions in cost and solvent waste generation, as well as improvements in assay quality and transferability.

Evaluation of the relationship between a pharmaceutical compound's distribution coefficient, log D and adsorption loss to polypropylene in urine and CSF.

BACKGROUND: The distribution coefficient, D, is a physicochemical property used to determine the partitioning of compounds between aqueous and hydrophobic media at a given pH. RESULTS: A clear relationship was observed between the calculated pH-dependent distribution coefficient of six representative pharmaceutical probe compounds and their propensity to partition between a relatively hydrophobic polypropylene surface and the aqueous matrices, human urine or human cerebrospinal fluid (CSF). Compound log D cut-off values of 1.5 and 3.8 for urine and CSF, respectively, were determined using a threshold of less than 20% adsorption to the polypropylene surface. CONCLUSION: The ability to forecast the adsorption of a given compound to a polypropylene container with urine and CSF offers an effective means for screening potential issues and identifying when additional testing and corrective measures may need to be applied.

Overcoming ionization effects through chromatography: a case study for the ESI-LC-MS/MS quantitation of a hydrophobic therapeutic agent in human serum using a stable-label internal standard.

Significant electrospray ionization effects were observed for a hydrophobic analyte and its stable isotopically labeled internal standard (SIL IS) in human plasma extracts. Analyte and SIL IS were slightly offset in retention within the suppression region resulting in a differential suppression that biased calculated concentrations. Six abundant endogenous phospholipids were identified as potential contributors to ionization suppression. Chromatographic conditions were optimized using pH (relative to the log D of the analytes), to better resolve nearby phospholipids from the analyte and SIL IS and minimize ionization suppression. The successful validation of this method demonstrates the value of investigating minor chromatographic changes to remediate detrimental ionization effects without further altering extraction procedures.

Investigation of an on-line two-dimensional chromatographic approach for peptide analysis in plasma by LC-MS-MS.

Reversed phase and hydrophilic interaction chromatography (HILIC) were successfully coupled for the on-line extraction and quantitative analysis of peptides by ESI-LC-MS/MS. A total of 11 peptides were utilized to determine the conditions for proper focusing and separation on both dimensions. Minor modifications to the initial organic composition of the first reversed-phase dimension provided options between a comprehensive (generic) or more selective approach for peptide transfer to the second HILIC dimension. Ion-pairing with trifluoroacetic acid (TFA) provided adequate chromatographic retention and peak symmetry for the selected peptides on both C18 and HILIC. The resulting signal suppression from TFA was partially recovered by a post-column "TFA fix" using acetic acid yielding improvements in sensitivity. Minimal sample preparation aligned with standard on-line extraction procedures provided highly reproducible and robust results for over 300 sequential matrix injections. Final optimized conditions were successfully employed for the quantitation of peptide PTHrP (1-36) in rat K(3)EDTA plasma from 25.0 to 10,000 ng/mL using PTHrP (1-34) as the analog internal standard. This highly orthogonal two-dimensional configuration was found to provide the unique selectivity required to overcome issues with interfering endogenous components and minimize electrospray ionization effects in biological samples.

Reduction of in-source collision-induced dissociation and thermolysis of sulopenem prodrugs for quantitative liquid chromatography/electrospray ionization mass spectrometric analysis by promoting sodium adduct formation.

Six chromatographically resolved sulopenem prodrugs were monitored for their potential to undergo both in-source collision-induced dissociation (CID) and thermolysis. Initial Q1 scans for each prodrug revealed the formation of intense [Prodrug2 + H]+, [Prodrug2 + Na]+, [Prodrug + Na]+, and [Sulopenem + Na]+ ions. Non-adduct-associated sulopenem ([Sulopenem + H]+) along with several additional lower mass ions were also observed. Product ion scans of [Prodrug3 + Na]+ showed the retention of the sodium adduct in the collision cell continuing down to opening of the beta-lactam ring. In-source CID and temperature experiments were conducted under chromatographic conditions while monitoring several of the latter ion transitions (i.e., adducts, dimers and degradants/fragments) for a given prodrug. The resulting ion profiles indicated the regions of greatest stability for temperature and declustering potential (DP) that provided the highest signal intensity for each prodrug and minimized in-source degradation. The heightened stability of adduct ions, relative to their appropriate counterpart (i.e., dimer to dimer adduct and prodrug to prodrug adduct ions), was observed under elevated temperature and DP conditions. The addition of 100 microM sodium to the mobile phase further enhanced the formation of these more stable adduct ions, yielding an optimal [Prodrug + Na]+ ion signal at temperatures from 400 to 600 degrees C. A clinical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for sulopenem prodrug PF-04064900 in buffered whole blood was successfully validated using sodium-fortified mobile phase and the [PF-04064900 + Na]+ ion for quantitation. A conservative five-fold increase in sensitivity from previously validated preclinical assays using the [PF-04064900 + H]+ precursor ion was achieved.