Gradient HPLC System Research Articles

Characterization of a Novel M4 PAM PET Radioligand [11C]PF06885190 in Nonhuman Primates (NHP).

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer's disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around -10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.

RP-HPLC Method Development and Validation for the Estimation of Midostaurin in Bulk and Pharmaceutical Dosage Form

An elementary, Valid, speedy and decisive strategy was developed to determine Midostaurin quantitatively in a fixed dosage form. Effective Chromatographic separation of Midostaurin was achieved by using Hypersil C18 Column (250 mm X 4.6 mm internal diameter, five μm particle size) using a mobile phase composed of Methanol and Buffer in the proportion of 75:25(by volume). The Mobile phase was siphoned using a gradient HPLC system at a flow rate of 1.0 ml/min, and quantification was based on peak area measurement at 270 nm. RT (Retention Time) for Midostaurin was 2.142 min, and dimensionality of Midostaurin was found to be linear with a statistic value of 0.999. The acceptance criteria of precision were relative variance should be less than 2.0%, and also the strategy showed precision 0.3 for Midostaurin, which shows that the tactic was precise. The full Recovery was found to be 99.96 %. Detection Limit and Quantitation Limit values for Midostaurin were found as 0.439 & 1.466. The exactness and authenticity were assessed by evaluation of validation parameters like linearity, precision, specificity, accuracy, LOD, LOQ values as per ICH guidelines. The proposed strategy has been applied to the formulation without additives interference and specific for the estimation of Midostaurin.

SIMULTANEOUS DETERMINATION OF ROSMARINIC ACID, SALVIANOLIC ACID B AND CAFFEIC ACID IN SALVIA LERIIFOLIA BENTH. ROOT, LEAF AND CALLUS EXTRACTS USING A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY DETECTION TECHNIQUE

Salvia leriifolia Benth. (Lamiaceae) is a medicinal plant with analgesic, hypoglycemic and anti-inflammatory properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. The present study aimed to determine the amounts of rosmarinic acid, salvianolic acid B and caffeic acid in the roots, leaves and calli of S. leriifolia using a simple, rapid and efficient HPLC method. In addition, the effect of different combinations of 2,4-dichlorophenoxyacetic acid and kinetin–as growth regulators–on phenolic composition was evaluated. The results indicated that the 3 phenolic acids were well separated using the applied gradient HPLC system [C18 (5mm) column; phosphoric acid 1% and methanol as mobile phase]. Limit of detection and quantification values for rosmarinic acid, caffeic acid and salvianolic acid B were 0.2 and 0.5, 1.0 and 2.0, and 0.5 and 1.0mg/mL, respectively. Inter- and intra-day variations for rosmarinic acid, caffeic acid and salvianolic acid B were 2.8% and 3.4%, 2.4% and 3.5%, and 4.8% and 4.7%, respectively. Recovery values for the 3 phenolic acids ranged from 96.3% to 98.9%. Rosmarinic acid was the most frequent phenolic acid in the ethanolic extract of roots, leaves and calli of S. leriifolia (0.33, 4.65 and 6.39 mg/g dry extract, respectively) followed by salvianolic acid B (0.11, 0.13 and 0.10 mg/g) and caffeic acid (0.15, 0.20 and 0.07 mg/g). The highest dry weight (0.226 g) of callus was achieved in medium containing 2 mg/L 2,4-D and 1 mg/L KIN. To conclude, the present study indicated that rosmarinic acid is the major phenolic acid in the roots, leaves and calli of S. leriifolia, using a simple, rapid, efficient and reproducible HPLC method for the separation and simultaneous determination of phenolic acids. Moreover, the content of evaluated phenolics was found to be higher in the leaf extract vs. root and callus extracts of the plant.

Stability-Indicating RP-HPLC Method for the Determination of Ambrisentan and Tadalafil in Pharmaceutical Dosage Form.

A simple, rapid, and highly selective RP-HPLC method was developed for the simultaneous determination of Ambrisentan (AMB) and Tadalafil (TADA) drug substances in the fixed dosage strength of 10 mg and 40 mg, respectively. Effective chromatographic separation was achieved using a Hypersil GOLD C18 column (150 mm × 4.6 mm internal diameter, 5 μm particle size) with a mobile phase composed of methanol, water, and acetonitrile in the ratio of 40:40:20 (by volume). The mobile phase was pumped using a gradient HPLC system at a flow rate of 0.5 mL/min, and quantification of the analytes was based on measuring their peak areas at 260 nm. The retention times for Ambrisentan and Tadalafil were about 2.80 and 7.10 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 1–20 μg/mL for Ambrisentan and 4–80 μg/mL for Tadalafil with correlation coefficients >0.990. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from the forced degradation (hydrolysis, oxidation, and photolysis) products. The validated HPLC method was successfully applied to the analysis of AMB and TADA in pharmaceutical dosage form.

Validated Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Azelnidipine and Olmesartan in Their Combined Dosage Form.

A simple, rapid, and highly selective RP-HPLC method was developed for the simultaneous determination of Azelnidipine (AZL) and Olmesartan (OLM) drug substances in the fixed dosage strength of 16 mg and 20 mg, respectively. Effective chromatographic separation was achieved using a Hypersil GOLD C18 column (150 mm × 4.6 mm internal diameter, 5 µm particle size) with a mobile phase composed of methanol, acetonitrile, and water in the ratio of 40:40:20 (by volume). The mobile phase was pumped using a gradient HPLC system at a flow rate of 0.5 mL/min, and quantification of the analytes was based on measuring their peak areas at 260 nm. The retention times for Azelnidipine and Olmesartan were about 8.56 and 3.04 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection, and quantification limits. Calibration curves were linear in the ranges of 2–48 μg/mL for Azelnidipine and 2.5–60 μg/mL for Olmesartan with correlation coefficients >0.990. The proposed method proved to be selective and stability-indicating by the resolution of the two analytes from the forced degradation (hydrolysis, oxidation, and photolysis) products. The validated HPLC method was successfully applied to the analysis of AZL and OLM in their combined dosage form.

Quantification of fatty acid oxidation products using online high-performance liquid chromatography tandem mass spectrometry

Oxidized fatty acids formed via lipid peroxidation are implicated in pathological processes such as inflammation and atherosclerosis. A number of methods may be used to detect specific oxidized fatty acids containing a single or multiple combinations of epoxide, hydroxyl, ketone, and hydroperoxide moieties on varying carbon chain lengths from C8 up to C30. Some of these methods are nonspecific and their use in biological systems is fraught with difficulty. Measures of specific oxidized fatty acid derivatives help in identifying oxidation pathways in pathological processes. We used liquid chromatography coupled with electrospray ionization tandem mass spectrometry as an efficient, selective, and sensitive method for identifying and analyzing multiple specific fatty acid peroxidation products in human plasma and other biological matrices. We then distilled the essential components of a number of these analyses to provide an efficient protocol by which fatty acid oxidation products and their parent compounds can be determined. In this protocol, addition of a synthetic internal standard to the sample, followed by base hydrolysis at elevated temperature and liquid–liquid phase sample extraction with lighter-than-water solvents, facilitates isolation of the oxidized fatty acid species. These species can be identified and accurately quantified using stable-isotope dilution and multiple-reaction monitoring. Use of a coupled multiplexed gradient HPLC system on the front end enables high-throughput chromatography and more efficient use of mass spectrometer time.

Direct method for quantification of glycidol fatty acid esters in edible oils

AbstractIn response to great concerns about glycidol fatty acid esters (GEs), trace contaminants in edible oils which possibly form during refining processes, we previously developed direct methods to quantify GEs using a combination of double SPE and HPLC‐MS. In this study, those quantitative methods were improved to be more rapid without the use of harmful chloroform. To improve the speed, the HPLC‐MS measuring time was reduced from 15 to 5 min by using a step gradient HPLC system, and the evaporation time after the first SPE was shortened from 4 to 1.5 h by changing the solvent used from acetonitrile to methanol. To replace chloroform with other solvents, we adopted tert‐butyl methyl ether/ethyl acetate to dissolve the oils and n‐hexane/ethyl acetate to separate them during the second SPE. The limit of quantification for GE‐in‐oil was 0.082–0.11 µg/g, which is the most sensitive of all existing methods. The recovery values of spiked GEs at concentrations of 1 or 10 µg/g were close to 100%. The levels quantified using an internal standard C17:0‐GE were comparable with those using external standards in four different edible oils tested. This rapid method not using chloroform can be useful as a routine method to quantify GEs in oils.

Characterisation of tannin‐containing herbal drugs by HPLC

Herbal drugs containing tannins are characterised in the European Pharmacopoeia by their tannin content analysed by the old, nonspecific, colorimetric Folin-Ciocalteu method. The result of the analysis is a single figure relating to the content of tannins, but this does not provide much information on the identity or status of the herbal drug. In the present paper methods for obtaining more detailed information of the constituents in these herbal drugs are described. The methods developed are based on a reversed-phase gradient HPLC system coupled to DAD, fluorescence, electrochemical and MS detectors. The HPLC system developed provides characteristic fingerprints of the herbal drugs when using UV detection at 250 nm. The fingerprints may be used for identification of tannin-containing herbal drugs. Methanolysis of the herbal drug generated methyl gallate and ellagic acid, which were analysed in the HPLC system. The molar ratio between methyl gallate and ellagic acid may also be used for the identification of herbal drugs. An HPLC system equipped with selective detectors was shown to be valuable in the identification of herbal tannin. Most promising was fingerprinting using UV detection, but methanolysis followed by HPLC also proved useful.

Application of .GAMMA.-Ray Irradiation to Reduction of Blank for Highly Sensitive Analysis of Low Molecular Weight Aldehydes in Water

For a highly sensitive analysis of formaldehyde and acetaldehyde in water, a degradation of formaldehyde and acetaldehyde in blank water by 60Co γ-ray irradiation was demonstrated. Formaldehyde and acetaldehyde in water were evaluated by using a large-volume injection/column switching/high pressure gradient HPLC system coupled with 2,4-dinitrophenylhydrazine derivatization. By γ-ray irradiation, formaldehyde was decomposed to formic acid, and the resulting formic acid was furthermore decomposed. Acetaldehyde also oxidized to acetic acid upon exposure to γ-rays. In the early stage of γ-ray irradiation for Milli-Q water, the formaldehyde and acetaldehyde in Milli-Q water increased, and many unknown peaks appeared in HPLC chromatograms. The formaldehyde, acetaldehyde and unknown species formed upon γ-ray irradiation may be derived from trace organic mater contained in Milli-Q water. Acetaldehyde and unknown peaks disappeared upon further γ-ray irradiation, and the formaldehyde peak was also diminished. In this report, purification of the DNPH derivatization reagent is also mentioned.

Analysing the Chemical Composition Distribution of Ethylene‐Acrylate Copolymers: Comparison of HT‐HPLC, CRYSTAF and TREF

AbstractHT‐HPLC is an attractive technique to analyse CCD of olefin copolymers and offers the possibility to shorten analysis times compared to fractionation techniques based on crystallisation. We have found that HT‐HPLC enables the selective elution of EMA and EBA copolymers according to their content of the polar co‐monomer. This fractionation could be confirmed by coupling the gradient HPLC system with FT‐IR spectroscopy. The CCDs obtained by this new method were then compared to the results from CRYSTAF. Both methods, HT‐HPLC and CRYSTAF, can discriminate between sets of samples having a narrow or a broad CCD. They can also prove the presence of acrylate‐poor fractions.magnified image

Relationship of serum carotenoids with the risk of breast cancer in Korean women

The relationship of serum antioxidant vitamin concentration and the risk of breast cancer was investigated in a case-control study in Korea. This study was carried out with 389 subjects consisting of 160 breast cancer patients and 229 control subjects from June 1994 to July 1995. The serum concentrations of α-tocopherol, carotenoids and retinol were measured simultaneously by a reverse phase, gradient HPLC system. Average serum concentrations of α-tocopherol, β-carotene and retinol were 7.63 ± 0.42 ug/ml, 40.5 ± 2.05 ug/dl, and 49.0 ± 4.10 ug/dl for cases and 11.0 ± 0.73 ug/ml, 48.3 ± 1.59 ug/dl and 64.6 ± 1.95 ug/dl for controls, respectively. Serum levels of α-tocopherol, β-carotene, cryptoxanthin, lutein+zeaxanthin, lycopene and retinol of breast cancer patients were significantly lower than those of controls. Odds ratios (ORs) adjusted for age, body mass index, smoking habit and alcohol drinking revealed a significant gradient of decreasing risk of breast cancer with increasing serum antioxidant vitamins. There were significant interactions between menopausal status and serum levels of antioxidant vitamins. The adjusted odds ratios for highest quintile compared with lowest quintile were 0.41 (95% CI, 0.18, 0.93) for α-tocopherol, 0.33 (95% CI, 0.15, 0.73) for β-carotene, 0.13 (95% CI, 0.06, 0.31) for zeaxanthin+lutein in premenopausal women; 0.13 (95% CI, 0.03, 0.66) for α-tocopherol, 0.28 (95% CI, 0.07, 1.12) for β-carotene, 0.12 (95% CI, 0.03, 0.58) for zeaxanthin+lutein in postmenopausal women. This results support inverse association between serum antioxidant vitamins and the risk of breast cancer in Korea and indicate that antioxidant vitamins may protect against breast cancer.

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: Application in human myocardium

Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- s-indacene-3-pentanoyl)-1-hexadecanoyl- sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 μg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.

Determination of carbohydrate-deficient transferrin in human serum using the Bio-Rad %CDT by HPLC test

Background Carbohydrate-deficient transferrin (CDT) in serum is a biomarker used to identify individuals with sustained, heavy alcohol consumption. This study evaluated the performance of a new commercial method for CDT, the Bio-Rad %CDT by HPLC test, that measures relative amounts of separate transferrin glycoforms in proportion to total transferrin. Method The samples used were two human serum pools (low/high disialotransferrin), 150 clinical sera with low to highly elevated disialotransferrin values, and 18 genetic transferrin variants. Glycoforms are separated on a gradient HPLC system, followed by specific measurement of the iron-transferrin complex at 460 nm. Comparison was made with an HPLC candidate reference method on an Agilent 1100 LC system. Results The Bio-Rad %CDT by HPLC test allowed for reproducible separation and quantification of the transferrin glycoforms within ∼ 6 min. Genetic variants were readily identified. For the low and high serum pool, the total CV was 8.5% and 4.3%, respectively. The relative amounts of disialotransferrin, the main CDT glycoform, were in good agreement with the results of the HPLC candidate reference method ( r 2 = 0.998, p < 0.0001). Conclusions The present results demonstrated that the Bio-Rad %CDT by HPLC test is appropriate for confirmatory and routine %CDT testing in human serum, with the added advantage over previously published HPLC methods of an improved serum sample pretreatment and a shorter total analysis time.

Electrospray[+] tandem quadrupole mass spectrometry in the elucidation of ergot alkaloids chromatographed by HPLC: screening of grass or forage samples for novel toxic compounds

Ergot alkaloids are mycotoxins generated by grass and grain pathogens such as Claviceps, for example. Ergot alkaloid-poisoning syndromes, such as tall fescue toxicosis from endophyte-infected tall fescue grass, are important veterinary problems for cattle, horses, sheep, pigs and chickens, with consequent impact on food, meat and dairy industries. Damage to livestock is of the order of a billion dollars a year in the United States alone. HPLC with UV and fluorescence detection are the predominant means of ergot alkaloid determination, with focus on quantitation of the marker compound ergovaline, although ELISA methods are undergoing investigation. These techniques are excellent for rapid detection, but of poor specificity in defining new or poorly characterized ergot alkaloids and related compounds. This paper demonstrates the facility of using electrospray(+) mass spectrometry with multiple reaction monitoring (MRM) detection during chromatographic examination of ergot alkaloid standards of lysergic acid, lysergol, ergonovine, ergovaline, ergotamine, ergocornine, ergocryptine and ergocrystine by HPLC. Ergoline-8 position epimers could be separated on the gradient HPLC system for ergocornine, ergocrystine and ergonovine and appeared as shoulders for ergotamine and ergovaline; epimers generally showed different patterns of relative intensity for specific MRM transitions. There was reasonable correspondence between retention of standards on the 2-mm ESI(+)MS phenyl-hexyl-based reverse phase column and those on the 4-mm C18-based column. Since up to 10% of clinical cases involving toxin exposure display unidentified chromatographic peaks, 11 samples of feed components associated with such cases were studied with developed MRM methods to attempt elucidation of crucial components if possible. Ergotamine appeared in all, ergovaline appeared in five and ergocornine appeared in six; ergonovine, ergocryptine, ergocrystine and lysergol also appeared in several. In addition, molecular weights of compounds newly revealed by mass spectrometry suggested ergosine, ergostine and ergoptine in four samples, for which standards were not available. Dehydrated products of ergotamine, ergocrystine and ergocornine were discovered, along with dihydrogenated ergocrystine and ergocryptine in seven of the samples, and the issue was raised as to whether dehydration was strictly an instrument-derived artifact. Finally, five of the samples, along with fescue seed standard, evidenced one or more of 14 new ergot alkaloids ranging in size from 381 to 611 molecular weight and with key mass spectral characteristics of ergot alkaloids, specifically the pair of peaks m/z 223 and 208, corresponding to the ergoline ring system and its demethylated variant, respectively. It is anticipated that findings such as these will provide impetus to future development of analytical methodology for these heretofore relatively rare ergot alkaloid species.

On-chip temperature gradient interaction chromatography

This paper reports the first integrated microelectromechanical system (MEMS) HPLC chip that consists of a parylene high-pressure LC column, an electrochemical sensor, a resistive heater and a thermal-isolation structure for on-chip temperature gradient interaction chromatography application. The separation column was 8 mm long, 100 μm wide, 25 μm high and was packed with 5 μm sized, C18-coated beads using conventional slurry-packing technique. A novel parylene-enhanced, air-gap thermal isolation technology was used to reduce heater power consumption by 58% and to reduce temperature rise in the off-column area by 67%. The fabricated chip consumed 400 mW when operated at 100 °C. To test the chromatography performance of the fabricated system, a mixture of derivatized amino acids was chosen for separation. A temporal temperature gradient scanning from 25 to 65 °C with a ramping rate of 3.6 °C/min was applied to the column during separation. Successful chromatographic separation of derivatized amino acids was carried out using our chip. Compared with conventional temperature gradient HPLC system which incorporates “macro oven” to generate temporal temperature gradient on the column, our chip's thermal performance, i.e., power consumption and thermal response, is greatly improved without sacrificing chromatography quality.

Separation, pre‐concentration, and HPLC analysis of methylxanthines in urine samples

An SPE method, using RP18 phases, for the simultaneous extraction of caffeine, theobromine, theophylline, paraxanthine, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, and 1,3,7-trimethyluric acid from urine has been developed. Besides a gradient HPLC system for the analysis of the compounds of interest on a LiChrosorb RP-18 (7 microm) column with mobile phase containing 0.05% aq. solution of trifluoroacetic acid and acetonitrile has been elaborated. The procedure has been successfully applied to the analysis of methylxanthines and methyluric acids in urine of patients with chronic asthma treated with theophylline and in urine of healthy subjects.

Determination of 4-amino- m-cresol and 5-amino- o-cresol and metabolites in human keratinocytes (HaCaT) by high-performance liquid chromatography with DAD and MS detection

A multi-step gradient HPLC system combined with DAD and MS detection has been developed for the determination of the oxidation hair dyes 4-amino- m-cresol (4-AC) and 5-amino- o-cresol (5-AC) and their metabolites in the alternative testing system human keratinocytes (HaCaT) cell culture. The culture medium induced by 3-methylcholanthrene (3-MC) was fortified with 4-AC or 5-AC and incubated for 24 h at 37 °C in order to produce metabolites. After several pre-cleaning steps, further cleaning was done by solid-phase extraction using C18 phenyl cartridges. Optimizing chromatographic conditions, a hybrid-based RP8 column was most suitable for the separation of the metabolites formed in HaCaT. Only one conjugation product, the N-acetylated derivative, could be identified for both 4-AC and 5-AC by LC/DAD/MS. The ionisation technique used for MS analysis was Atmospheric Pressure Ionization (API).

Characterization of eptifibatide during drug formulation stability assays

The objective of the study was to determine the identity of a new impurity detected in HPLC chromatograms of research samples of eptifibatide manufactured by a new process and formulated into drug product. The identification of the unknown impurity was required in order to understand the mechanism of its formation. The analysis was performed by using tandem mass spectrometers coupled with a reversed-phase gradient HPLC system. The unknown compound was then structurally elucidated by matrix-assisted laser desorption ionization (MALDI) tandem mass spectrometry. The mass spectrometric results showed that the protonated molecular ion of the unknown compound was m/ z 862.3347 with molecular formula: C 36H 52N 11O 10S 2. The unknown compound was a linear peptide and was related to Asp-clipped eptifibatide. It was formed from Asp-clipped eptifibatide by the reaction of the amino group of tryptophan moiety with formaldehyde followed by electrophilic attack on the nitrogen of indole.

Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression.

We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis. The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests. The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components. Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30. However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Comparison between liquid chromatography–UV detection and liquid chromatography–mass spectrometry for the characterization of impurities and/or degradants present in trimethoprim tablets

The characterization of impurities and/or degradants present in pharmaceutical compounds is an important part of the drug development process. Although LC–UV is commonly employed for impurities and degradant compound determination, LC–MS techniques are proposed in this work to be a viable modern alternative for the characterization of these compounds. LC–UV and LC–MS were compared for the detection of impurities present in different brands of trimethoprim tablets by using an in-line LC–UV–MS system with atmospheric pressure chemical ionization source (APCI) coupled with a reversed-phase gradient HPLC system. It was shown that, although chemical noise was higher when using full-scan LC–MS compared to LC–UV, low level impurities were better detected by mass spectrometry (MS) when modern software algorithms are employed. These included the “Contour” chromatogram algorithm and/or the “component detection algorithm” (CODA). In addition, MS allowed for the simultaneous determination of the molecular masses and some structural information of the impurities and/or degradants. The results also showed a large difference in the purity of trimethoprim among different manufacturers. LC–MS and tandem MS techniques were employed to acquire fragmentation patterns for trimethoprim and its degradants to gain insight into their structures.