Degradation of a pharmaceutical in HPLC grade methanol containing trace level formaldehyde.

An anomalous peak was observed in the HPLC/UV analysis of a developmental drug product. High resolution LC/MS revealed that the mass of this degradant was 12 Da greater than the drug substance, corresponding to a net gain of a single carbon atom. The degradant was reproduced by incubating the drug substance with formaldehyde, followed by isolation using normal phase chromatography and structure elucidation by NMR. It was determined to be an analytical artifact caused by the nucleophilic reaction of the drug substance with trace levels of formaldehyde in the methanol diluent. Typical formaldehyde levels in various grades of methanol were determined, leading to the adoption of spectrophotometric purity solvent to mitigate the recurrence of this artifact. This work demonstrates that even ppm levels of impurities in solvents can cause significant degradation of drug product and the HPLC grade solvents are not always suitable for HPLC analysis in drug product development.

Strategies for the analysis of highly reactive pinacolboronate esters.

Pinacolboronate esters (or boronic acid, pinacol esters) are widely used in the Suzuki coupling reaction to connect organic building blocks for the total synthesis of complex molecules. The 2-aminopyrimidine-5-pinacolboronate ester was used as a starting material in the synthesis of a development compound, necessitating a chromatographic purity method to assess its quality. This aryl pinacolboronate ester posed unique analytical challenges due to its facile hydrolysis to the corresponding boronic acid, which is nonvolatile and poorly soluble in organic solvents. This made GC and normal-phase HPLC analysis unsuitable. In reversed-phase mode, typical sample preparation and analysis conditions promoted rapid sample degradation to the boronic acid. To overcome these challenges, unconventional approaches were necessary in order to stabilize 2-aminopyrimidine-5-pinacolboronate ester, adequately solubilize its boronic acid, and produce acceptable separation and retention. The final method employed non-aqueous and aprotic diluent, and a reversed-phase separation using highly basic mobile phases (pH 12.4) with an ion pairing reagent. These strategies were successfully applied to several other reactive pinacolboronate esters for purity analysis, demonstrating broad applicability to this unique class of compounds.

Characterization and resolution of reversed phase HPLC chromatography failure attributed to sulfobutylether-beta-cyclodextrin in a pharmaceutical sample preparation.

A reversed phase HPLC method developed for a drug product formulation using hydroxypropyl-beta-cyclodextrin (HPCD) was rendered ineffective for analyzing a similar formulation containing sulfobutylether-beta-cyclodextrin (SBECD). The active pharmaceutical ingredient (API) and the majority of its impurities became more strongly retained, eluting as an incoherent conglomerate of peaks. Furthermore, this phenomenon was reproduced in subsequent injections of the API reference standard. Based on HPLC and LC-ESI-MS studies, the chromatography failure was attributed to the accumulation of SBECD on the HPLC column. The subsequent interaction of the API with bound SBECD resulted in the aberrant chromatography. An anion-exchange solid-phase extraction treatment was developed and qualified to selectively remove SBECD from sample solutions, thereby allowing the same HPLC method to be used. The sample treatment procedure exhibited suitable accuracy and precision for quantitating the API and its impurities, and resulted in typical chromatographic profiles.

Analysis of monoethanolamine by derivatization with Marfey's reagent and HPLC.

A sensitive and selective method for determining the residual monoethanolamine in a developmental drug substance is developed and validated. Marfey's reagent, which is commonly used for the chiral analysis of amino acids, is reacted with the primary amine group of monoethanolamine and then analyzed by high-performance liquid chromatography-UV at 340 nm. Quantitation is performed by a standard addition method by preparing drug substance samples with added monoethanolamine ranging from 0.25-1.0 microg/mL (equivalent to 12.5-50 ppm with respect to the drug substance). The method performance is evaluated for linearity, specificity, detection and quantitation limits, accuracy, precision, and sample stability. The method is linear from 0.25-1.0 microg/mL with a coefficient of determination (r(2)) > 0.95. The accuracy and precision obtained is 105.5 +/- 4.8% (n = 3). The limits of detection and quantitation are 0.03 and 0.10 microg/mL, respectively. Instrument precision (% relative standard deviation of six injections of a derivatized 0.5 microg/mL monoethanolamine solution on two separate days) is >/= 2.0%. This method is suitable for the determination of monoethanolamine at the 25 ppm level in drug substance.

Multiresidue determination of pesticides in malt beverages by capillary gas chromatography with mass spectrometry and selected ion monitoring.

A method was developed to determine pesticides in malt beverages using solid phase extraction on a polymeric cartridge and sample cleanup with a MgSO4-topped aminopropyl cartridge, followed by capillary gas chromatography with electron impact mass spectrometry in the selected ion monitoring mode [GC-MS(SIM)]. Three GC injections were required to analyze and identify organophosphate, organohalogen, and organonitrogen pesticides. The pesticides were identified by the retention times of peaks of the target ion and qualifier-to-target ion ratios. GC detection limits for most of the pesticides were 5-10 ng/mL, and linearity was determined from 50 to 5000 ng/mL. Fortification studies were performed at 10 ng/mL for three malt beverages that differ in properties such as alcohol content, solids, and appearance. The recoveries from the three malt beverages were greater than 70% for 85 of the 142 pesticides (including isomers) studied. The data showed that the different malt beverage matrixes had no significant effect on the recoveries. This method was then applied to the screening and analysis of malt beverages for pesticides, resulting in the detection of the insectide carbaryl and the fungicide dimethomorph in real samples. The study indicates that pesticide levels in malt beverages are significantly lower than the tolerance levels set by the United States Environmental Protection Agency for malt beverage starting ingredients. The use of the extraction/cleanup procedure and analysis by GC-MS(SIM) proved effective in screening malt beverages for a wide variety of pesticides.

The extraction and infrared identification of gamma-hydroxybutyric acid (GHB) from aqueous solutions.

The chemical analysis of gamma-hydroxybutyric acid (GHB) in most forensic laboratories is complicated by the highly polar nature of the GHB molecule, which makes it unsuitable for direct analysis by gas chromatography (GC). Consequently, a popular analytical approach is to convert GHB into the corresponding lactone or a derivative compound that is then identified by mass spectrometry employed in conjunction with GC (GC/MS). An alternative approach is presented here where GHB may be isolated as a free acid specie from complex aqueous solutions employing a liquid-liquid extraction technique. This approach can yield a relatively pure residue of GHB that presents an infrared transmission spectrum that is sufficiently distinct for identification purposes. Infrared spectroscopy (IR) is a very popular technique that is available to most crime laboratories. The liquid-liquid extraction behavior of GHB is examined in detail and the uniqueness of the infrared spectrum is discussed.

Multiresidue pesticide analysis in wines by solid-phase extraction and capillary gas chromatography-mass spectrometric detection with selective ion monitoring.

A method was developed to determine pesticides in wines. The pesticides were extracted from the wine using solid-phase extraction on a polymeric cartridge, and the coextractives were removed with an aminopropyl-MgSO(4) cartridge. Analysis was performed using capillary gas chromatography with electron impact mass spectrometric detection in selective ion monitoring mode (GC-MSD/SIM). Three injections are required to analyze all 153 organohalogen, organonitrogen, organophosphate, and organosulfur pesticides and residues. Pesticides were confirmed by retention times of the target ions and three qualifier-to-target ion ratios. Detection limits for most of the pesticides were less than 0.005 mg/L, and quantitation was determined from approximately 0.01 to 5 mg/L. Spike recoveries were performed by fortifying red and white wines at 0.01 and 0.10 mg/L. At the 0.01 ppm level, the spike recoveries were greater than 70% for 116 and 124 pesticides (out of 153) in red and white wines, respectively, whereas at the higher spike concentration of 0.10 mg/L, the recoveries were greater than 70% for 123 and 128 pesticides in red and white wines, respectively. The recoveries of less than 70% were most likely from pesticide polarity or lability, resulting in the inefficient adsorption of the pesticide to the polymeric sorbent, ineffective elution of the pesticide from the sorbent, or thermal degradation of the pesticide under GC-MSD conditions.

Photoreduction of the chloropropionic acid of carfentrazone-ethyl in sodium sulfide [corrected].

The purpose of this study was to determine whether the conversion of carfentrazone-chloropropionic acid to carfentrazone-propionic acid in sunlit rice paddies is attributed to photoreduction. Model solutions (Na2S with quinoids) irradiated by laboratory ultraviolet light dechlorinated carfentrazone-chloropropionic acid (1.6-28.4% yield of carfentrazone-propionic acid), though Na2S alone was also reactive. Minor conversion (0-2.5%) occurred in the dark, along with dehydrochlorination to carfentrazone-cinnamic acid. Carfentrazone-propionic acid formed proportionally to the Na2S concentration (0-50.7% at 1.3-91 mM), whereas acidic pH inhibited reactivity. Photoreduction with Na2S was verified with 2-chloropropionic acid conversion to propionic acid (53.5%) and by minor 4-chlorophenoxyacetic acid dechlorination. Dissolved Na2S was the primary photoreductant, whereas reduced quinones degraded carfentrazone-chloropropionic acid by an alternate reaction. A survey of ambient rice field conditions indicates that carfentrazone-chloropropionic acid photoreduction is not directly attributed to H2S/HS- in this environment, though reduced quinones may be involved to an unknown extent.