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  • 1. Abuzooda, Thana
    et al.
    Amini, Ahmad
    Swedish Drug Agency,751 03 Uppsala, Sweden.
    Abdel-Rehim, Mohamed
    Graphite-based microextraction by packed sorbent for online extraction of β-blockers from human plasma samples2015In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 992, p. 86-90Article in journal (Refereed)
    Abstract [en]

    In the present work a new graphitic material (Carbon-XCOS) was used as a sorbent for microextraction by packed sorbent (MEPS). The β-blockers metoprolol and acebutolol in plasma samples were extracted and detected online using Carbon-MEPS syringe and liquid chromatography and tandem mass spectrometry (LC-MS/MS). Factors affecting the MEPS performance such as conditioning, washing and elution solutions were investigated. The validation of the bioanalytical method was performed using human plasma. The standard curve ranged from 10 to 2000nM and the lower limit of quantification (LLOQ) was set to 10nM. The method validation showed good accuracy and precision for the quality control (QC) samples at three concentration levels (30, 800 and 1600nM). The accuracy values of the QC samples were in the range of 86-108% (n=18). The precision values of intra- and inter-day for QC samples ranged from 4.4% to 14.4% (RSD) for the both studied analytes. The coefficient of determination (R(2)) values were ≥0.999 (n=3).

  • 2.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Analysis of somatropin by double-injection capillary-zone electrophoresis in polybrene/chondroitin sulfate a double-coated capillaries2016In: Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols / [ed] Nguyet Thuy Tran, Myriam Taverna, New York: Springer-Verlag New York, 2016, p. 93-105Chapter in book (Refereed)
    Abstract [en]

    Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter- plug distance. Here, the principle for the separation of somatropin charge variants is described.

  • 3.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Double-injection capillary electrophoresis for the identification of analytes2014In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 35, no 20, p. 2915-2921Article in journal (Refereed)
    Abstract [en]

    This paper presents a new approach for identifying analytes by CE. The compound to be identified is analyzed together with the corresponding reference standard during a double injection capillary electrophoretic run. The inter-plug distance is regulated by applying an electrical field over the capillary for a predetermined time period (tPE). The migration time of an analyte being exposed to the partial electrophoresis was calculated from the partial migration time (tmig(p)) as described in this paper. The identification is based on the closeness of agreement between the calculated migration time (tmig(c)) and observed migration time (tmig) of the reference standard. The validity of the derived equations was checked by analyzing several substances such as caffeine, melamine, acetyl salicylic acid, paracetamol, ibuprofen, metoprolol, naproxen, somatropin, several insulin analogs, as well as different pharmaceutical and natural products. The migration time ratios for the identified solutes varied between 0.996 and 1.006 (i.e., 1.001 ± 0.005), indicating good agreement between the observed and calculated migration times.

  • 4.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Identification of Ɛ-Caprolactam and Melamine in Polyvinyl- Pyrrolidone Powder by Double Injection Micellar Elektrokinetic Chromatography2015In: Pharmaceutica analytica acta, ISSN 2153-2435, Vol. 6, no 11, article id 1000442Article in journal (Refereed)
    Abstract [en]

    A double-injection micellar electrokinetic chromatography (DIMEKC) method for the identification of Ɛ- caprolactam andmelamine deliberately added to povidone (polyvinylpyrrolidone) products has been developed. The separations were performedin 89 mM phosphate buffer (pH 7.4) containing 52 mM sodium dodecyl sulfate (SDS) in fused silica capillaries with UV absorptiondetection at 200 nm. The identification relied on the agreement between the calculated migration time (t mig(c) ) of the analytes andthe migration time (t mig ) of their corresponding reference standards being analysed simultaneously within a double injection run.The migration time of the analytes was calculated from the partial migration times (t mig(p) ) as described in this paper. The migrationtime ratios (t mig(c) / t mig ) varied

  • 5.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Identification of ε-caprolactam, melamine and urea in polyvinylpyrrolidone powders by micellar electrokinetic chromatography2014In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 91, p. 12-16Article in journal (Refereed)
    Abstract [en]

    A sodium dodecyl sulfate micellar electrokinetic chromatography (SDS-MEKC) method for the simultaneous separation and identification of ɛ-caprolactam, melamine and urea deliberately added to polyvinylpyrrolidone (povidone) products has been developed. All samples to be analyzed contained paracetamol as an internal marker (IM). The optimized separations were performed in 50 mM phosphate buffer (pH 7.0) containing 2% (w/v) sodium dodecyl sulfate (SDS) in fused silica capillaries with UV absorption detection at 200 nm. The method was validated with respect to repeatability and intermediate precision, selectivity and robustness with satisfactory results. The relative migration times (RMT) were found to be between 0.03% and 0.13% for intra-day precision and between 0.50% and 0.60% for inter-day precision in four days. The detection limits were determined to be 1.3 (11.5 μM), 0.4 (3.5 μM) and 41 μg/ml (0.4 mM) for ɛ-caprolactam, melamine and urea, respectively.

  • 6.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Separation of somatropin charge variants by multiple-injection CZE with Polybrene/chondroitin sulfate A double-coated capillaries2013In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 36, no 16, p. 2686-2690Article in journal (Refereed)
    Abstract [en]

    The performance of dynamic double-coated fused-silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused-silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30 degrees C. The coating significantly reduced the interactions between the proteins and the surface of the fused-silica capillary. The first five separations performed in a new bare fused-silica capillary were discarded because of very poor separation performance as a result of protein-surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD 6.5%) in the double-coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD 0.3%) determined by using uncoated and coated capillaries.

  • 7.
    Amini, Ahmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Lodén, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Principles for different modes of multiple-injection CZE2008In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 19, p. 3952-3958Article in journal (Refereed)
    Abstract [en]

    This paper introduces four different modes of multiple-injection CZE (MICZE). The validity of these MICZE models was evaluated by the experimental data. Prior to the application of MICZE, the electrophoretic conditions are developed in the single-injection mode by adjusting different experimental parameters such as pH, type and concentration of buffer additives and temperature. Based on the migration time difference (tmig) between the analyte and the internal standard or injection marker, one or more MICZE modes can be employed. The injection marker is added to the sample to compensate for injection-volume fluctuations. The inter-plug distance is regulated by applying an electrical field over the capillary for a short period of time between each injection. After the final injection, the separation is completed by electrophoresis for a time period corresponding to that in the single-injection mode

  • 8.
    Amini, Ahmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Nilsson, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Quantitative analysis of polypeptide pharmaceuticals by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry2008In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 46, no 3, p. 411-417Article in journal (Refereed)
    Abstract [en]

    An accurate method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been developed for quantitative analysis of calcitonin and insulin in different commercially available pharmaceutical products. Tryptic peptides derived from these polypeptides were chemically modified at their C-terminal lysine-residues with 2-methoxy-4,5-dihydro-imidazole (light tagging) as standard and deuterated 2-methoxy-4,5-dihydro-imidazole (heavy tagging) as internal standard (IS). The heavy modified tryptic peptides (4D-Lys tag), differed by four atomic mass units from the corresponding light labelled counterparts (4H-Lys tag). The normalized peak areas (the ratio between the light and heavy tagged peptides) were used to construct a standard curve to determine the concentration of the analytes. The concentrations of calcitonin and insulin content of the analyzed pharmaceutical products were accurately determined, and less than 5% error was obtained between the present method and the manufacturer specified values. It was also found that the cysteine residues in CSNLSTCVLGK from tryptic calcitonin were converted to lanthionine by the loss of one sulfhydryl group during the labelling procedure.

  • 9.
    Amini, Ahmad
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Rundlöf, Torgny
    Rönnquist, Kerstin
    Tydén, Monica
    Turunen, Taina
    Korhola, Paula
    Anette, Perolari
    A protocol for the quality assessment of illegally distributed human growth hormones with respect to identity, purity, endotoxin level and microorganism content2015In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 7, no 20, p. 8857-8864Article in journal (Refereed)
    Abstract [en]

    Different methods based on MALDI-TOF-MS and double injection capillary zone electrophoresis (DICZE) were used for the identification and purity determination of somatropin in illegally distributed products. During the past few years, more than 200 products suspected to contain somatropin have been analysed. Some of the samples were also subjected to control microorganisms and endotoxins. The identification of somatropin was carried out by peptide mapping using trypsin as the proteolytic enzyme. A double chain peptide cross-linked via a disulfide bond was used as the signature peptide. Capillary electrophoresis in double injection mode was applied to both identification and purity determination of the samples. The identification was based on the comparison between the observed migration time of the reference standard and the calculated migration time of the analyte, being present in the second injection plug. The DICZE provides electrophoretic fingerprints of intact somatropin and the related proteins which facilitate the identification. In addition, some of these samples revealed the presence of microorganisms as well as a high level of endotoxins. Taken together, the doubtful quality of the analysed samples and the microbiological findings represent a serious threat for the consumers and public health.

  • 10. Durgaryan, Andranik
    et al.
    Rundlöf, Torgny
    Lavén, Martin
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Identification of human chorionic gonadotropin hormone in illegally distributed products by MALDI-TOF mass spectrometry and double-injection capillary zone electrophoresis2016In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 8, no 21, p. 4188-4196Article in journal (Refereed)
    Abstract [en]

    This paper describes two complementary methods, based on peptide-mass fingerprinting (PMF) using MALDI-TOF mass spectrometry and double injection capillary zone electrophoresis for identification of human chorionic gonadotropin (hCG). The identification was performed by determining the amino acid composition and comparing measured mass spectra with those predicted in silico. Capillary zone electrophoresis in double injection mode (DICZE) was used to confirm the identity of the protein. In DICZE, the identification was performed by analyzing illegal samples together with the corresponding reference standard during a double injection capillary zone electrophoretic run. The identification was based on the closeness of agreement between the calculated migration time (t(mig(c))) and the observed migration time (tmig) of the reference standard. Furthermore, the DICZE method provides the possibility to compare the electrophoretic separation patterns of the native reference standard and the target analyte. The inner surface of the capillary was dynamically coated with putrescine, present in the BGE at 8.2 mM, to improve the separation. The CZE analyses revealed that the protein consisted of at least ten glycosylated isoforms.

  • 11. Elmongy, Hatem
    et al.
    Ahmed, Hytham
    Wahbi, Abdel-Aziz
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry. Swedish Drug Agency, Uppsala, Sweden.
    Colmsjö, Anders
    Abdel-Rehim, Mohamed
    Determination of metoprolol enantiomers in human plasma and saliva samples utilizing microextraction by packed sorbent and liquid chromatography-tandem mass spectrometry2016In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 30, no 8, p. 1309-1317Article in journal (Refereed)
    Abstract [en]

    A sensitive, accurate and reliable bioanalytical method for the enantioselective determination of metoprolol in plasma and saliva samples utilizing liquid chromatography-electrospray ionization tandem mass spectrometry was developed and validated. Human plasma and saliva samples were pretreated by microextraction by packed sorbent (MEPS) prior to analysis. A new MEPS syringe form with two inputs was used. Metoprolol enantiomers and internal standard pentycaine (IS) were eluted from MEPS sorbent using isopropanol after removal of matrix interferences using aliquots of 5% methanol in water. Complete separation of metoprolol enantiomers was achieved on a Cellulose-SB column (150 × 4.6 mm, 5 μm) using isocratic elution with mobile phase 0.1% ammonium hydroxide in hexane-isopropanol (80:20, v/v) with a flow rate of 0.8 mL/min. A post-column solvent-assisted ionization was applied to enhance metoprolol ionization signal in positive mode monitoring (+ES) using 0.5% formic acid in isopropanol at a flow rate of 0.2 mL/min. The total chromatographic run time was 10 min for each injection. The detection of metoprolol in plasma and saliva samples was performed using triple quadrupole tandem mass spectrometer in +ES under the following mass transitions: m/z 268.08 → 72.09 for metoprolol and m/z 303.3 → 154.3 for IS. The linearity range was 2.5-500 ng/mL for both R- and S-metoprolol in plasma and saliva. The limits of detection and quantitation for both enantiomers were 0.5 and 2.5 ng/mL respectively, in both matrices (plasma and saliva). The intra- and inter-day precisions were presented in terms of RSD values for replicate analysis of quality control samples and were <5%; the accuracy of determinations varied from 96 to 99%. The method was able to determine the therapeutic levels of metoprolol enantiomers in both human plasma and saliva samples successfully, which can aid in therapeutic drug monitoring in clinical laboratories.

  • 12.
    Lodén, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Quantification of Buserelin in a Pharmaceutical Product by Multiple-injection CZE2007In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 28, no 10, p. 1548-1556Article in journal (Refereed)
    Abstract [en]

    An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 μug/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 ± 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).

  • 13.
    Lodén, Henrik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Pettersson, Curt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Arvidsson, Torbjörn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis2008In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1207, no 1-2, p. 181-185Article in journal (Refereed)
    Abstract [en]

    A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30 kV) over the capillary for different time periods, i.e., tPE1 and tPE2. The samples in each set were isolated from each other by partial electrophoresis for 2.35 min (tPE1), while the sample sets were separated for 10.50 min (tPE2). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8 mg salbutamol. The average salbutamol content was determined to 7.8 mg (±0.3 mg) from simultaneous analyses of the 15 different tablets.

  • 14.
    Nilsson, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Amini, Ahmad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Analytical Pharmaceutical Chemistry.
    Wretlind, Bengt
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin: Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin2007In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 856, no 1-2, p. 75-80Article in journal (Refereed)
    Abstract [en]

    Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.

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