Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL-1, 0.28 ng mL-1, 0.50 ng mL-1, 0.41 ng mL-1 and 0.91 ng mL-1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00-400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Peptide-N-glycosidase F or A treatment and procainamide-labeling for identification and quantification of N-glycans in two types of mammalian glycoproteins using UPLC and LC-MS/MS.

BACKGROUND: N-glycans in glycoproteins can affect physicochemical properties of proteins; however, some reported N-glycan structures are inconsistent depending on the type of glycoprotein or the preparation methods. OBJECTIVE: To obtain consistent results for qualitative and quantitative analyses of N-glycans, N-glycans obtained by different preparation methods were compared for two types of mammalian glycoproteins. METHODS: N-glycans are released by peptide-N-glycosidase F (PF) or A (PA) from two model mammalian glycoproteins, bovine fetuin (with three glycosylation sites) and human IgG (with a single glycosylation site), and labeled with a fluorescent tag [2-aminobenzamide (AB) or procainamide (ProA)]. The structure and quantity of each N-glycan were determined using UPLC and LC-MS/MS. RESULTS: The 21 N-glycans in fetuin and another 21 N-glycans in IgG by either PF-ProA or PA-ProA were identified using LC-MS/MS. The N-glycans in fetuin (8-13 N-glycans were previously reported) and in IgG (19 N-glycans were previously reported), which could not be identified by using the widely used PF-AB, were all identified by using PF-ProA or PA-ProA. The quantities (%) of the N-glycans (>0.1 %) relative to the total amount of N-glycans (100 %) obtained by AB- and ProA-labeling using LC-MS/MS had a similar tendency. However, the absolute quantities (pmol) of the N-glycans estimated using UPLC and LC-MS/MS were more efficiently determined with ProA-labeling than with AB-labeling. Thus, PF-ProA or PA-ProA allows for more effective identification and quantification of N-glycans than PF-AB in glycoprotein, particularly bovine fetuin. This study is the first comparative analysis for the identification and relative and absolute quantification of N-glycans in glycoproteins with PF-ProA and PA-ProA using UPLC and LC-MS/MS.

Flow method based on liquid-liquid extraction using deep eutectic solvent for the spectrofluorimetric determination of procainamide in human saliva.

In the current study, liquid-liquid extraction, using deep eutectic solvent (DES) as a "green" extraction solvent, was coupled with a stepwise injection system for the first time. The suggested approach was applied for the development of spectrofluorimetric method for procainamide determination. The method is based on aspiration of saliva sample and DES (choline chloride with glycerol at a 1:2M ratio) solution into the mixing chamber of a flow system, followed by injection of acetonitrile into the mixed DES-sample solution. The extraction process and final phase separation were then promoted by air-bubbling. After phase separation, the DES phase, containing the extracted procainamide, was transported to a spectrofluorimetric detector. The excitation and emission wavelengths were set at 280nm and 347nm, respectively. The calibration plot was linear in the range of 5×10-6 to 5×10-5molL-1. The limit of detection, calculated as 3σ of a blank test (n=10), was found to be 1.5×10-6molL-1. The developed method was successfully applied for the determination of procainamide in human saliva samples, and the analytical results agreed rather well with the results obtained by the reference HPLC-UV method.

Simultaneous determination of four local anesthetics by CE with ECL and study on interaction between procainamide and human serum albumin.

A new method of capillary electrophoresis (CE) coupled with tris(2, 2'-bipyridyl) ruthenium(II) electrochemiluminescence (ECL) detection has been developed to detect four local anesthetics procainamide (PAH), tetracaine (TCH), proparacaine (PCH) and cinchocaine (CIN) simultaneously. An europium (III)-doped prussian blue analogue film (Eu-PB) modified platinum electrode was prepared and applied to improve the detection sensitivity. The parameters including additives, concentration and pH of the running buffer, separation voltage and detection potential that affect CE separation and ECL detection were optimized in detail. The four local anesthetics were baseline separated and detected within 10min under the optimized conditions. The detection limits (LOD) of PAH, TCH, PCH and CIN are 5.5×10(-8), 9.6×10(-8), 2.5×10(-8) and 3.5×10(-8)molL(-1) (S/N=3), respectively. RSDs of the migration time for four analytes range from 1.2% to 2.5% within intraday and from 2.4% to 4.9% in interday, RSDs of the peak area for four analytes are from 1.7% to 3.3% within intraday and from 2.2% to 5.6% in interday, respectively. The limits of quantitation (LOQ) (S/N=10) for PAH, TCH, PCH and CIN in human urine sample are 5.9×10(-7), 9.2×10(-7), 8.3×10(-7) and 5.0×10(-7)molL(-1), separately. The recoveries (n=3) of four analytes in human urine are from 87.6% to 107.7% with less than 5.9% in RSDs. The developed method was used to determine four local anesthetics in human urine samples and investigate the interaction between PAH and human serum albumin (HSA). The number of binding sites and the binding constant of PAH with HSA were calculated to be 1.03 and 2.4×10(4)Lmol(-1), respectively.

Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds.

In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 µmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 µmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Potentiometric immunosensor using artificial antibody based on molecularly imprinted polymers.

A potentiometric artificial immunosensor based on a molecularly imprinted polymer was prepared as a detecting element in micro total analysis systems with the intent of providing easy clinical analysis. As the structure and transducing mechanism of this sensor are very simple, construction of a single microsensor should be quite easy. Multimicrosensor arrays applicable to several kinds of analytes will be attainable by both changing the template molecule to be imprinted and reducing the sensor size. The response characteristics of this sensor were evaluated by measuring the response potential to serotonin, which was used as a model material. The obtained sensor was highly responsive to serotonin in water but not to tryptamine, acetaminophen, or procainamide. This phenomenon confirms that the sensor recognizes serotonin and that it functions as a specific artificial immunosensor. Quick measurement is possible because the response time, defined as the time required to achieve 95% of the magnitude of the equilibrated signal, correspond to approximately 12 s. The sensor's determination and detection limits were found to be 1 mumol/L and 100 pmol/L, respectively. These results suggest that our strategy can be applied to construction of a potentiometric artificial immunosensor.

Stacking for nonaqueous capillary electrophoresis.

Nonaqueous capillary electrophoresis (NACE) is a useful mode in CE for separation and quantification of hydrophobic compounds. However, because of the low conductivity of most of the organic solutions, stacking is not used often in this technique and the sample volume is very limited. As a result of the small sample volume, the detection limits are poor. Furthermore, NACE is affected greatly by the presence of salts in the sample. Here, we show that transient isotachophoresis (t-ITP) can be used easily in this type of electrophoresis to enhance the detection limits and also to reverse the deleterious effects of salts in the sample. Several factors, which affect the stacking in this type of electrophoresis, are described. For example, the presence of salts in the organic solvent, type of sample introduction, and the solvent for the terminating ion were all found to have profound effects on the degree of concentration. Furthermore, the separation time can be shortened by t-ITP.

[The detection and quantitative determination of lidocaine and novocainamide in biological fluids]. / Obnaruzhenie i kolichestvennoe opredelenie lidokaina i novokainamida v biologicheskikh zhidkostiakh.

Offers methods for isolation of lidocaine and novocainamide present together in the urine, blood plasma, and saliva. Demonstrates the possibility of identifying these substances in biological fluids by thin-layer and gas liquid chromatography. Proposes methods for measuring lidocaine and novocainamide in biological objects by UV spectrometry (combined polynomial) and gas liquid chromatography. Demonstrates the reliability of the results.

Direct determination of procainamide and N-acetylprocainamide by capillary zone electrophoresis in pharmaceutical formulations and urine.

In this work a new sensitive capillary zone electrophoresis method for the direct determination of procainamide (PA) and N-acetylprocainamide (NAPA) in pharmaceutical formulations and urine samples without any extraction and/or preconcentration steps has been developed. The determination was carried out in a fused-silica capillary of 43.5 cm (35.9 cm length to the detector) x 0.75 micron J.D. Phosphate 0.05 M buffer was used as the background electrolyte and 10 kV separation voltage was applied. The separation of PA and NAPA is possible in a wide range of pH from 1.7 to 9.7. However, in order to avoid the effect of the urine matrix, it is optimal to work at pH 7.7. The determination of PA and NAPA takes less than 5 min while high resolution is achieved. The detection limits obtained, 1.235 micrograms/ml and 0.359 microgram/ml for PA and NAPA respectively, are lower than those for GC method normally reported.

Development of a novel octadecyl-bonded silica column and evaluation of its reliability in chromatographic analysis.

The chromatographic characterization of an octadecyl-bonded silica (ODS) column for high-performance liquid chromatography is described. In general, columns of the same type but obtained from various manufacturers give different chromatographic results, due to differences in the purity of column packings, the properties of the silica gel supports and the density of silanols on the surface of the silica gel. In order to solve this problem and to obtain a high performance ODS column, a series of methods with different samples and conditions were evaluated. The result is important for the optimization of conditions for the synthesis of ODS packings and for minimizing the deleterious effects arising from adsorption activity and metal impurity.

Chromatographic (TLC) analysis of diltiazem in pharmaceutical form in presence of other antiarrhythmics.

Diltiazem was identified by TLC method on silica gel by ascending technique, in presence of five another antiarrhythmic drugs (disopyramide, flecainide, lidocaine, lorcainide, procainamide). Suitable conditions for separation of diltiazem were established using ethanol-benzene-dioxane-ammonia (2:20:16:3) as the mobile phase. When using acidified iodoplatinate solution or Dragendorff reagent as indicators, the amount of diltiazem as small as 100 ng can be identified. The TLC method is simple and sensitive and it was used to identify diltiazem in tablets.

Use of 7,7,8,8-tetracyanoquinodimethane for spectrophotometric determination of certain local anaesthetics and procainamide hydrochloride.

A simple and rapid spectrophotometric procedure has been developed for the determination of four local anaesthetics containing a free primary amine moiety and of procainamide hydrochloride as the drug substances and in dosage forms. The method is based on the reaction of the drug with 7,7,8,8-tetracyanoquinodimethane in alkaline solution to produce yellow products. The chromogen was measured at 473 nm. The effects of several variables on colour development were established. Job's plots of absorbance versus molar ratio of drug to reagent indicated a 1:1 ratio for all the drugs studied. Results of the analysis of drug substances and their dosage forms by the proposed method are in good agreement with those obtained by the USP XX method.

[Ion pair HPLC determination of p-aminobenzoic acid as an impurity in procaine and procainamide hydrochlorides]. / Determinazione dell'acido p-amino-benzoico quale impurezza nei cloridrati di procaina e procainamide mediante HPLC a coppia ionica.

A simple, specific and sensitive high-performance liquid chromatographic method for the determination of p-amino benzoic acid (PABA) as impurity in procaine and procainamide hydrochlorides has been developed. The compounds were chromatographed on reversed phase C-18 using water-methanol-acetonitrile solvent system containing sodium lauryl sulphate ion-pair reagent.

Algunas consideraciones sobre la valoración de procainamida clorhidrato / Appraisement of procainamide hydrochloride

Se estudian métodos y alternativas para la determinación del contenido de procainamida clorhidrato, materia prima. Se realiza un análisis acerca de la influencia del anhídrico acético cuando se valora este fármaco anhidrovolumétricamente y se recomienda el método que de acuerdo con los ensayos efectuados y su evaluación estadística demostró ser el más eficiente, preciso, sencillo y rápido

Analysis of drug in tissue homogenates by high performance liquid chromatography with direct injection and column switching.

An HPLC method with direct sample injections and column switching was investigated for the analysis of drugs in tissue homogenates. The appropriate precolumn packings and analytical column packings were surveyed in order to obtain quantitative recovery. The use of a large bore end-fitting filter for the precolumn avoided the interference due to minute tissue particles. A minicolumn was used to trap the analyte or clean up the sample. The method was applicable to hydrophilic as well as hydrophobic drugs in liver, kidney or heart tissues, and has been extensively used in the determination of drugs in centrifugal cell fractions such as the nucleus, mitochondria and microsome.

Stability of procainamide hydrochloride in neutralized 5% dextrose injection.

The stability of procainamide hydrochloride in neutralized 5% dextrose injection was studied. Sixty-four admixtures were prepared by adding either 2 mL (for 0.4% admixtures) or 4 mL (for 0.8% admixtures) of procainamide hydrochloride to 250 mL of 5% dextrose injection in plastic containers. The pH of 32 of these admixtures (16 of each type) was adjusted to 7.5. These 32 admixtures represented the neutralized group, and the remaining 32 represented the control group. The admixtures were stored at either 23-25 degrees C (room temperature) or 5 degrees C (refrigeration) for 24 hours. Procainamide hydrochloride concentrations in each sample were determined by high-performance liquid chromatography immediately after the admixtures were prepared and at various intervals during storage. Procainamide concentrations decreased over time in 5% dextrose injection. The decrease was significantly less for admixtures in neutralized 5% dextrose injection, those stored under refrigeration, and those with an 0.8% concentration of drug. Decreases in procainamide hydrochloride concentrations in the control admixtures might have been caused by procainamide-dextrose complexation. Initial concentrations of procainamide hydrochloride in 5% dextrose injection can be adequately maintained over a 24-hour storage period by neutralizing the 5% dextrose injection or storing the admixture at 5 degrees C. However, because it is impractical to maintain the necessary temperature condition during a 24-hour infusion, neutralization might be the most viable alternative when extended stability of procainamide hydrochloride in 5% dextrose injection is required.

Stability of milrinone and digoxin, furosemide, procainamide hydrochloride, propranolol hydrochloride, quinidine gluconate, or verapamil hydrochloride in 5% dextrose injection.

The stability of milrinone and digoxin, furosemide, procainamide hydrochloride, propranolol hydrochloride, quinidine gluconate, or verapamil hydrochloride in 5% dextrose injection containing milrinone was studied. Milrinone admixtures with digoxin, furosemide, propranolol hydrochloride, quinidine gluconate, and verapamil hydrochloride were studied at two concentrations. Admixtures of milrinone and procainamide hydrochloride were studied at four concentrations. Duplicate solutions of each admixture and each control were prepared and stored in glass containers for four hours at room temperature (22-23 degrees C), under normal fluorescent lights. The samples were analyzed immediately by visual inspection, tested for pH, and assayed by high-performance liquid chromatography (HPLC). Milrinone 0.35 mg/mL-furosemide 4 mg/mL and milrinone 0.1 mg/mL-furosemide 5 mg/mL admixtures precipitated immediately after preparation and were not studied by HPLC. No changes in pH or visual appearance were observed in the remaining admixtures after storage at room temperature for four hours. Admixtures containing milrinone 0.175 or 0.2 mg/mL and procainamide hydrochloride 1, 2, or 4 mg/mL satisfied the USP standard for procainamide hydrochloride injection USP assay after one hour but failed this test in all cases after four hours. No degradation of milrinone was observed in any of the admixtures containing procainamide hydrochloride. Milrinone and furosemide are incompatible in 5% dextrose injection and should be administered separately. The remaining admixtures were compatible, and all except those containing procainamide hydrochloride were stable for four hours at room temperature.

More-sensitive enzyme-multiplied immunoassay technique for procainamide and N-acetylprocainamide in plasma, serum, and urine.

A commercially available (Syva Co.) enzyme-multiplied immunoassay technique (EMIT) for the quantitative determination of procainamide (PA) and N-acetylprocainamide (NAPA) was modified to allow automated quantitative analysis of approximately 100 samples per day, in a working range of 0.1 to 2.0 micrograms/mL. Such a test was needed to evaluate the pharmacokinetic characteristics of controlled-release dosage forms characterized by long half-lives at low plasma concentration. Analytical recovery of PA and NAPA from serum, plasma, and urine was satisfactory, but at extreme ratios for PA:NAPA the accuracy of determining the lower-concentration component became unsatisfactory. In fact, however, we found no such ratios in 5400 clinical samples assayed by this procedure.

Reversed-phase liquid chromatography method for measurement of procainamide and three metabolites in serum and urine: percent of dose excreted as deethyl metabolites.

We report a reversed-phase high-performance liquid chromatography method for the determination of procainamide (PA) and three of its metabolites, n-acetylprocainamide (NAPA), deethylprocainamide (DEPA), and deethyl-n-acetylprocainamide (DENAPA), in serum and urine. (p-Amino)-n-(2-dipropylaminoethyl)-benzamide was the internal standard. A phenyl column (1.0-mL/min flow rate) and a mobile phase consisting of 0.075 M acetate buffer (pH 4.3):acetonitrile (20:3) resulted in a total chromatography time of 21.6 min. The optimum detector wavelength was 270 nm. Maximum linear concentrations were 37.8, 34.2, 20.0, and 16.3 mg/L for DEPA, DENAPA, PA, and NAPA, respectively. Minimum detectable concentrations were 0.05 mg/L or less for all four compounds. One-tenth milliliter of sample was extracted into methylene chloride:2-propyl alcohol (9:1). Extraction efficiencies were independent of concentration or biological fluid for each compound. Standard curves were linear and best-fit by dividing the curve into two portions and/or using weighted linear regression. Within-day and day-to-day precison were excellent. No interfering substances were observed in the serum or urine of normal subjects with the exception of caffeine, which was resolved by alteration of the mobile phase. Advantages of this method include a small sample volume, low minimum detectable concentrations, and an order of elution which enhances the detectability of the deethyl metabolites. Urinary excretion of DEPA and DENAPA accounted for an average of only 0.58 and 0.53%, respectively, of PA doses administered intravenously to six normal volunteers.