Benzoyl Chloride Derivatization Research Articles

Gas chromatography mass spectrometer determination of dimethylamine impurity in N,N-dimethylformamide solvent by derivatization of N,N-dimethylbenzamide with benzoyl chloride agent.

This study describes the development of a reliable and linear analytical method for precisely determining dimethylamine impurity in N,N-dimethylformamide solvent utilizing a benzoyl chloride derivatization reagent and a gas chromatography mass spectrometer. Benzoyl chloride was used to derivatize dimethylamine. At normal temperature, benzoyl chloride combined with dimethylamine, producing N,N-dimethylbenzamide. This method separated N,N-dimethylbenzamide using Rtx-5 amine (30 m × 0.32 mm × 1.50 μm) as the stationary phase, helium as the carrier gas, argon as the collision gas, and methanol as the diluent. The column flow rate was 2mL/min. The retention time of N,N-dimethylbenzamide was determined to be 8.5min. Precision, linearity, and accuracy were tested using ICH Q2 (R2) and USP<1225> guidelines. The percentage coefficient of variation (CV) for N,N-dimethylbenzamide in the system suitability parameter was 1.1%. The correlation coefficient of N,N-dimethylbenzamide was found to be >0.99. In the method precision parameter, the % CV for N,N-dimethylbenzamide was found to be 1.9%, whereas the % CV for N,N-dimethylbenzamide was 1.2% in intermediate precision. The percentage recovery of N,N-dimethylbenzamide was determined to be between 80% and 98%.

Benzoyl Chloride Derivatization Advances the Quantification of Dissolved Polar Metabolites on Coral Reefs.

Extracellular chemical cues constitute much of the language of life among marine organisms, from microbes to mammals. Changes in this chemical pool serve as invisible signals of overall ecosystem health and disruption to this finely tuned equilibrium. In coral reefs, the scope and magnitude of the chemicals involved in maintaining reef equilibria are largely unknown. Processes involving small, polar molecules, which form the majority components of labile dissolved organic carbon, are often poorly captured using traditional techniques. We employed chemical derivatization with mass spectrometry-based targeted exometabolomics to quantify polar dissolved phase metabolites on five coral reefs in the U.S. Virgin Islands. We quantified 45 polar exometabolites, demonstrated their spatial variability, and contextualized these findings in terms of geographic and benthic cover differences. By comparing our results to previously published coral reef exometabolomes, we show the novel quantification of 23 metabolites, including central carbon metabolism compounds (e.g., glutamate) and novel metabolites such as homoserine betaine. We highlight the immense potential of chemical derivatization-based exometabolomics for quantifying labile chemical cues on coral reefs and measuring molecular level responses to environmental stressors. Overall, improving our understanding of the composition and dynamics of reef exometabolites is vital for effective ecosystem monitoring and management strategies.

High-performance liquid chromatography determination of polyamine contents in poultry based on precolumn benzoyl chloride derivatization

Polyamines are widely present in animal cells and perform important physiological and biochemical functions. Moderate intake of polyamines contributes to good health. Poultry is one of the common sources of polyamines in daily life. However, different tissues from poultry contain different amounts of polyamine. In this study, an efficient and accurate method for the determination of polyamines was developed by high-performance liquid chromatography (HPLC) with precolumn benzoyl chloride derivatization. The linearity of the polyamines was good between 10–400 nmol/mL, with the limit of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 0.37 and 1.23 nmol/mL. The recoveries of the method ranged from 94.73 % to 106.58 % with high accuracy (RSD < 7 %) and no significant matrix effects or interferences. The RSD of intra-day, inter-day and inter-batch repeatability in the range of 0.07–1.57 %, 0.08–1.03 % and 1.46–4.57 %. The method was successfully used to detect polyamines in poultry tissue, with putrescine, spermidine and spermine levels ranging from 3.24 to 29.85 mg/kg, 0.46 to 164.11 mg/kg and 63.39 to 489.57 mg/kg, respectively. Polyamine levels were higher in the spleen, thymus and bursa of fabricius and lower in the heart and muscle. Furthermore, the method was employed to study the effect of exogenous spermine on polyamine deposition in different tissues of geese. In summary, the method is suitable for the detection of polyamine in animal tissues.

Development of a quantification method for arginase inhibitors by LC-MS/MS with benzoyl chloride derivatization

Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 μm, 30 × 3 mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1→176.1, 439.4→181.2, 334.9→150.0 and 339.9→150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00–10,000 ng/mL with QC values of 5, 50 and 500 ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.

Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes

LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e., specific bases), other hydroxyl groups may be derivatized too. Previous studies have examined BzCl concentration, reaction times, and reaction temperatures for derivatization of amines and phenols for LC-MS analysis; however, use of different bases, base concentration, and extending to conditions to hydroxyl groups for LC-MS analysis has not been well-studied. To address this understudied area and identify reaction conditions for both amino and hydroxyl groups, we performed a systematic study of reaction conditions on multiple classes of potential targets. For selected derivatization methods, detection limits and performance in a variety of biological matrices were assessed. Results highlight the importance of tailoring derivatization methods for a given application as they varied by molecule and/or molecule class. Compared to the standard BzCl method commonly used, alternative methods were identified to better derivatize challenging analytes (glucosamine, choline, cortisol, uridine, cytidine) with detection limits reaching 1100, 9, 38, 170, and 67 nM compared to undetectable, 170, 86, 1000, and 86 nM respectively. Sub-nanomolar detection limits were achieved for norepinephrine with alternative derivatization approaches. Improved derivatization methods for several classes and molecules including nucleosides, steroids, and molecules containing hydroxyl groups were also identified.

The Development of an Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry Method for Biogenic Amines in Fish Samples

Biogenic amines (BAs) are a group of substances that are formed from amino acids by decarboxylation or amination and transamination of aldehydes and ketones. They may have either an aliphatic, aromatic, or heterocyclic structure. Their quantity determines their effects and optimum amounts are essential for physiological functions, but excess BAs causes various toxic effects throughout the human body. In our study, to rapidly determine 14 BAs (histamine, tyramine, dopamine, tryptamine, serotonin, putrescine, spermine, spermidine, octopamine, benzylamine, 1-Phenylethanamine, cadaverine, 2-Phenethylamine, and agmatine) in real fish samples, an ultra-performance liquid chromatography-tandem mass spectrometry method was established. The fish sample was extracted by acetonitrile with 0.1% formic acid and stable biogenic amine derivatives could be obtained by benzoyl chloride derivatization with a shorter reaction time. The method showed good linearity with a linear range of 3-4 orders of magnitude and regression coefficients ranging from 0.9961 to 0.9999. The calculated LODs ranged from 0.1 to 20 nM and the LOQs ranged from 0.3 to 60 nM. Satisfactory recovery was obtained from 84.6% to 119.3%. The proposed method was employed to determine the concentration levels of biogenic amine derivatives in different fish. The results indicated that this method was suitable for the analysis of biogenic amines.

Small molecules released from islets of Langerhans determined by liquid chromatography - mass spectrometry.

Islets of Langerhans are the endocrine tissue within the pancreas that secrete hormones for maintenance of blood glucose homeostasis. A variety of small molecules including classical neurotransmitters are also released from islets. While the roles of most of these small molecules are unknown, some have been hypothesized to play a critical role in islet physiology. To better understand their role on islet function, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantify 39 small molecules released from islets. Benzoyl chloride derivatization of analyte molecules was used to impart retention and facilitate electrospray ionization efficiency. Separation was achieved on a 2.1 × 150 mm column packed with 2.7 μm core–shell C18 particles. Calibration curves showed excellent linearity between the concentration and analyte response, with relative standard deviations of the analyte responses below 15% and limits of detection from 0.01–40 nM. The method was applied to examine small molecules released from murine and human islets of Langerhans after static incubation and perfusion with glucose. Results showed a decrease in secretion rates with increasing glucose concentration for most of the analytes. Secretion rates were found to be higher in human islets compared to their murine counterpart. This method will be useful in understanding the roles of small molecules in biological systems.

Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).

Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.

A microfluidic chip for on-line derivatization and application to in vivo neurochemical monitoring.

Microfluidic chips can perform a broad range of automated fluid manipulation operations for chemical analysis including on-line reactions. Derivatization reactions carried out on-chip reduce manual sample preparation and improve experimental throughput. In this work we develop a chip for on-line benzoyl chloride derivatization coupled to microdialysis, an in vivo sampling technique. Benzoyl chloride derivatization is useful for the analysis of small molecule neurochemicals in complex biological matrices using HPLC-MS/MS. The addition of one or more benzoyl groups to small, polar compounds containing amines, phenols, thiols, and certain alcohols improves reversed phase chromatographic retention, electrospray ionization efficiency, and analyte stability. The current derivatization protocol requires a three-step manual sample preparation, which ultimately limits the utility of this method for rapid sample collection and large sample sets. A glass microfluidic chip was developed for derivatizing microdialysis fractions on-line as they exit the probe for collection and off-line analysis with HPLC-MS/MS. Calibration curves for 21 neurochemicals prepared using the on-chip method showed linearity (R2 > 0.99), limits of detection (0.1-500 nM), and peak area RSDs (4-14%) comparable to manual derivatization. Method temporal resolution was investigated both in vitro and in vivo showing rapid rise times for all analytes, which was limited by fraction length (3 min) rather than the device. The platform was applied to basal measurements in the striatum of awake rats where 19 of 21 neurochemicals were above the limit of detection. For a typical 2 h study, a minimum of 120 pipetting steps are eliminated per animal. Such a device provides a useful tool for the analysis of small molecules in biological matrices which may extend beyond microdialysis to other sampling techniques.

Broad targeted analysis of neurochemicals in rat serum using liquid chromatography tandem mass spectrometry with chemical derivatization.

In this study, an efficient and sensitive assay for the detection of 42 polar neurochemicals, including neurotransmitters, amino acids, and biogenic amines, was established by combining reversed-phase liquid chromatography tandem mass spectrometry with chemical derivatization. An optimally designed benzoyl chloride derivatization was easily conducted in a one-pot reaction and stable neurochemical derivatives were obtained under mild conditions within 5min (except for acetylcholine and melatonin). Derivatization also enabled the introduction of heavy labeling of the analytes through the use of labeled derivatization agents. Chromatography separation was performed on an HSS T3 column within 15min by gradient elution. Multiple reaction monitoring acquisition mode enabled quantitation of neurochemicals with limits of detection of 0.05 to 11.63nM and lower limits of quantitation of 0.09 to 46.50nM in rat serum. The assay was well validated in terms of linearity and extraction recovery. Furthermore, the instrumental precision, specificity, matrix effect, accuracy, precision, stability, dilution effect, and carry-over effect were also validated. Finally, the overall efficacy of the assay was experimentally tested using serum from six Sprague-Dawley rats. The results demonstrated that the developed method is effective for broad targeted analysis of 42 neurochemicals in serum.

LC-MS/MS determination of plasma catecholamines after selective extraction by borated zirconia.

For the first time, boronate affinity chromatography and metal oxide affinity chromatography mechanisms for cis-diol-compounds extraction are simultaneously realized on a single material, termed borated zirconia. This material was prepared by hydrolyzing zirconium butoxide with boric acid under non-aqueous environment. The diameter of formed particles is around 200nm. By extracting catechol under different pH conditions or with phosphate ion competition, the dual affinity mechanisms of borated zirconia are confirmed. Benefiting from such unique feature, borated zirconia can function well under neutral condition. By using borated zirconia for dispersive solid phase extraction, specific capture of cis-diol-containing catecholamines, including epinephrine (E), norepinephrine (NE) and dopamine (DA), was achieved. A reliable LC-MS/MS method was established and validated for quantification of these target analytes in plasma samples after derivatisation with benzoyl chloride. The linear ranges are 0.010-0.200ng·mL-1 for E and DA, and 0.050-1.000ng·mL-1 for NE. The limits of quantification are 0.008, 0.020 and 0.004ng·mL-1 for E, NE and DA respectively. By analyzing samples from healthy volunteers and schizophrenia patients, the plasma concentrations of E, NE and DA were found to behigher for the latter. Graphical AbstractSchematic representation of boronate combined metal oxide affinity chromatography (BMOAC) extraction of cis-diol catecholamines from human plasma by borated zirconia following with benzoyl chloride derivatization and LC-MS determination.

Ethylene glycol poisoning requiring critical care: Three case reports

Rapid diagnosis and treatment of ethylene glycol poisoning can be challenging and is critical for reducing the morbidity and the mortality of this intoxication. Cases from our center were retrospectively reviewed. From the 12 cases of ethylene glycol poisoning found since 2007, we present here three with significant toxicity and requiring critical care. Case 1 was an accidental intoxication in a 67 year-old male presenting dysarthria and altered level of consciousness. The patient mistakenly drank a bottle of beer containing a blue liquid 15 h before admission. Case 2 was a voluntary intoxication in a 56 year-old male with coma. The patient was found unconscious under a bridge with 9 wine bottles near him. An open bottle of car antifreeze was found at his home. Case 3 was a voluntary intoxication in a 46 year-old female presenting dyspnea. Ingestion amount was around 3 L of antifreeze 2 days before. Ethylene glycol plasma level was measured using liquid chromatography with ultraviolet detection after benzoyl chloride derivatization. At admission, case 1 showed metabolic acidosis (pH = 7.01) with very high serum lactate (> 20 mmol/L, acute renal failure (serum creatinine = 141 μmol/mL) and acute respiratory failure. The patient was transferred to an intensive care unit (ICU) where he was intubated and received renal replacement therapy (RRT). Ethylene glycol plasma level was 614 mg/L. Fomepizole was started 10 hours after admission. The patient was hospitalized for 35 days. Evolution was favorable but marked with an acute coronary syndrome. Cardiac function was recovered afterward. At admission in the ICU, case 2 was in a coma (Glasgow coma scale 3), and presented dyspnea, metabolic acidosis (pH = 6.90) with very high serum lactate (> 20 mmol/L), acute renal failure (serum creatinine = 141 μmol/mL) and hyperkaliemia (6.6 mmol/L). The patient was intubated and received RRT. Ethylene glycol plasma level was 402 mg/L. Fomepizole was started 18 h after admission. The patient was hospitalized for 21 days. Evolution was favorable except for a lower lip pressure ulcer and chronic kidney injury. Follow up at 2 months showed a partial recovery of renal function. Case 3 presented at admission metabolic acidosis (pH = 7.14) with high serum lactate (8.1 mmol/L), acute respiratory failure and acute renal failure (serum creatinine = 114 μmol/L). After transfer to the ICU, the patient was intubated and received RRT. Ethylene glycol plasma level was 607 mg/L. Fomepizole was started about 4 h after admission. The patient was hospitalized for 23 days. Evolution was marked by a ventilator-associated pneumonia but was favorable otherwise. These 3 cases have in common a severe acidosis associated with an acute renal failure and respiratory disorders. High serum lactate has to be interpreted with caution due to analytical interference [1] . Neurological toxicity was characterized for case 1 and 2. The long hospitalization duration (21 to 35 days) reflects the severity of the intoxications. Fomepizole antidote treatment must be started as soon as possible. Ethylene glycol plasma concentration should be obtained to confirm the diagnosis if available. Even though ethylene glycol poisoning is well known, cases are rare but nonetheless can currently be life-threatening. They require fast diagnostic and treatment to avoid morbidity [2] .

Quantification of three triterpenic acids in dried rosemary using HPLC-fluorescence detection and 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride derivatization.

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) and detected using HPLC-fluorescence (FL). Dried rosemary (50mg) was ground, added to 3ml of ethanol, sonicated for 40min, then the sample solution was added to a mixture of 1% trimethylamine and 1mM DIB-Cl in acetonitrile. The mixture was settled for 5min at room temperature, then the DIB-triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250×4.6mm, 6μm) eluted with 25mM acetate buffer (pH4.5)/methanol/acetonitrile (=8:10:82v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λex ) and 490nm (λem ) and the maximum retention time of the derivatives was 30min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r≥0.997) in the range 2.5-100ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.

A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with liquid chromatography-tandem mass spectrometry analysis.

Tetrahydrobiopterin (BH4) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH4 has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH4 by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH4-BC), ultra-sensitive and reliable quantification of endogenous BH4 in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH4-BC-d5 was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL-1. In the end, after investigation of plasma BH4 in healthy volunteers (n = 38), we found that the levels of BH4 were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH4-related fields.

Determination of cycloserine in microdialysis samples using liquid chromatography-tandem mass spectrometry with benzoyl chloride derivatization.

A new method for the analysis of cycloserine (4-amino-3-isoxazolidinone, CYC) in rat microdialysis samples has been developed. This method consists of derivatizing the CYC with benzoyl chloride, which transforms primary amines into highly stable derivatives. An attractive feature of this method was that the derivatization reaction is straightforward and can be completed within 10 min. The formed derivative, in contrast to the non-derivatized analyte, exhibited increased chromatographic retention and decreased matrix effects resulting from the co-elution of other components using reversed-phase liquid chromatography and on-line switching. Detection on a quadrupole-linear ion trap mass spectrometer (AB3200 Q-Trap) was performed using electrospray tandem mass spectrometry in multiple reaction monitoring mode. Various derivatization parameters were optimized in order to improve chromatographic separation and minimize ion suppression. In particular, the benzoylation reaction was improved to enhance the reproducibility and sensitivity of the chromatographic method. The transition m/z 207.1 → 105.1 was acquired to monitor the CYC derivatization products. The method was fully validated for its sensitivity, selectivity, matrix effect and stability. A good linearity over the selected range (r > 0.99, range = 22-2200 mg/L), as well as accuracy and precision within ±7% of the target values, was obtained. The assay described herein was successfully applied to quantitatively measure CYC in the lung and blood of anesthetized rats.

Determination of amines and phenolic acids in wine with benzoyl chloride derivatization and liquid chromatography–mass spectrometry

Amine and phenolic metabolites are important contributors to the flavor and health effects of many foods, including wine. Determination of these metabolites often involves UV detection following separation by liquid chromatography. While this is sufficient for some applications, chemical derivatization with LC–MS provides greater sensitivity and selectivity relative to LC-UV.We have developed an assay for 56 amine and phenolic metabolites in wine using benzoyl chloride derivatization and LC–MS. Isotopically labeled benzoyl chloride was used to prepare internal standards for each metabolite. Nanomolar limits of detection were achieved for all metabolites. To demonstrate the application of this assay, we compared metabolite profiles from Merlot and Cabernet Sauvignon wines from California and Australia. We found five metabolites which were significantly different when grouped by varietal, while twenty-four were different when grouped by location of production. This shows that the method can identify differences between various wines.

High-sensitivity detection of biogenic amines with multiple reaction monitoring in fish based on benzoyl chloride derivatization

In this study an efficient and sensitive method for biogenic amines detection was established by combining rapid extraction and derivatization with multiple reaction monitoring (MRM) based on ultra high performance liquid chromatography-triple quadrupole mass spectrometry. Bead-beating disruption and extraction using 5-sulfosalicylic acid not only improved the extraction efficiency but also was environmentally friendly. Benzoyl chloride derivatization could obtain stable biogenic amine derivatives with a shorter reaction time. By combining with MRM detection mode, higher detection sensitivity for biogenic amines could be achieved. The method showed a good linearity with linear range of 3–4 orders of magnitude and regression coefficients ranging from 0.9966 to 0.9999. The limit of detection and limit of quantitation could even reach lower pg/mL level. Satisfactory recovery was obtained from 74.9% to 119.3%. And the derivatives were stable within 48h at 4°C. The method established was used to determine content of biogenic amines in different fishes at different storage conditions. The results indicated that this method was suitable for analysis of biogenic amines.

Benzoyl chloride derivatization with liquid chromatography–mass spectrometry for targeted metabolomics of neurochemicals in biological samples

Widely targeted metabolomic assays are useful because they provide quantitative data on large groups of related compounds. We report a high performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS) method that utilizes benzoyl chloride labeling for 70 neurologically relevant compounds, including catecholamines, indoleamines, amino acids, polyamines, trace amines, antioxidants, energy compounds, and their metabolites. The method includes neurotransmitters and metabolites found in both vertebrates and insects. This method was applied to analyze microdialysate from rats, human cerebrospinal fluid, human serum, fly tissue homogenate, and fly hemolymph, demonstrating its broad versatility for multiple physiological contexts and model systems. Limits of detection for most assayed compounds were below 10nM, relative standard deviations were below 10%, and carryover was less than 5% for 70 compounds separated in 20min, with a total analysis time of 33min. This broadly applicable method provides robust monitoring of multiple analytes, utilizes small sample sizes, and can be applied to diverse matrices. The assay will be of value for evaluating normal physiological changes in metabolism in neurochemical systems. The results demonstrate the utility of benzoyl chloride labeling with HPLC–MS/MS for widely targeted metabolomics assays.

A validated LC-MS/MS method for neurotransmitter metabolite analysis in human cerebrospinal fluid using benzoyl chloride derivatization.

Human cerebrospinal fluid (CSF) is often acquired in Phase I clinical trials to assess the CNS penetration of new pharmacological agents and to search for biomarkers associated with PD effects. Robust methods for neurotransmitter metabolites in CSF have proven elusive, in part due to inadequate reversed phase LC retention. Benzoyl chloride derivatization was used to promote retention for LC-MS/MS for a panel of neurotransmitter metabolites while delivering a concise method for sample preparation. A validated assay in human CSF was obtained for 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3,4-dihydroxyphenylglycol and 5-hydroxyindoleacetic acid. This method is differentiated from other LC-MS/MS methods by delivering results in line with full regulatory expectations.

Development and Validation of a Specific RP-HPLC Method for the Estimation of γ-Aminobutyric Acid in Rat Brain Tissue Samples Using Benzoyl Chloride Derivatization and PDA Detection

A new, rapid, and specific reversed phase high-performance liquid chromatographic (RP-HPLC) method involving precolumn derivatization with benzoyl chloride was developed and validated for the estimation of γ-aminobutyric acid (GABA) in rat brain tissue preparations. The derivatization product of GABA was identified by melting point, infrared, and proton nuclear magnetic resonance (1H NMR) spectroscopy to be n-benzoyl GABA. Various parameters which influenced derivatization and elusion were optimized. The chromatographic system consisted of C-18 column with ultraviolet (UV)—photodiode array detection ranging from 210 to 400 nm. Elution with an isocratic mobile phase consisting of 0.025 M disodium hydrogen phosphate buffer—methanol (65:35, v/v; pH 6) at a flow rate of 1 mL min−1 yielded sharp and specific peak of n-benzoyl GABA within 7 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity, and stability, wherein the benzoyl derivative of GABA showed stab...