Compound 2β-carbomethoxy-3β-(4-chlorophenyl)tropane (β-CCT) is a key intermediate for the synthesis of some clinical dopamine transporter (DAT) imaging agents. Potential impurities from synthesis ...process of β-CCT and degradation during storage might have detrimental effect on the final imaging agents. Thus, it is necessary to guarantee the quality of β-CCT. In this study, a rapid, sensitive and accurate high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of β-CCT and its related substances. The chromatographic separation was achieved on a reverse-phase phenomenex™ Gemini C18 column with an isocratic mobile phase consisted of methanol, water and TFA (30:70:0.1 v/v/v). The flow rate was 1.0 mL/min at 30 °C and samples were monitored at 220 nm. The method was validated concerning system suitability, linearity, accuracy, precision, specificity, robustness and stability. The limit of detection (LOD) and the limit of quantification (LOQ) of β-CCT were 0.5 and 1.5 μg/mL, respectively. The linearity range of β-CCT was 1.5–450 μg/mL with a good linear correlation coefficient (R2 = 0.9999) between the peak response and concentration. Specificity investigation through forced degradation experiments displayed that β-CCT was stable in acidic, thermal and photolytic degradation conditions, but significantly unstable in alkaline and oxidative conditions. With the developed chromatographic method, possible impurity α-CCT from synthetic process and potential degradation products could be well separated from β-CCT. Good recovery and precision were manifested in the assay method. These results indicated that the present method would be suitable for not only the quality assurance of β-CCT in regular production sample assays but also the monitoring and determination of its related substances.
Calcitriol (1α,25-dihydroxyvitamin D3, 1), a classical vitamin D drug, is indicated primarily in the treatment of patients with postmenopausal osteoporosis and renal osteodystrophy. In this study, a ...practical synthesis of calcitriol (1), from readily available commercial vitamin D2 (5) via hub intermediate 18, has been accomplished in 9% overall yield. This semi-synthetic process embedded four prominent elements of vitamin D chemistry: (1) cheletropic sulfur dioxide (SO2) adduction for the isomerization of the characteristic triene from (5Z,7E) to (5E,7E), or for the protection of the triene for selective ozonolysis of the side chain, and cheletropic extrusion of SO2 from the adduct in ethanolic sodium bicarbonate to retrieve the triene; (2) direct, regio- and stereoselective 1α-hydroxylation of 3β-TBS-protected (5E)-calciferol intermediate 19 using selenium dioxide in the presence of N-methylmorpholine N-oxide as a re-oxidant in a hot mixture of methylene chloride and methanol; (3) nickel(0)-mediated conjugate addition of the 22-iodide 23 to electron-deficient ethyl acrylate followed by Grignard reaction with methylmagnesium bromide to construct the calcitriol side chain; and (4) triplet-sensitized photoisomerization of 26 to access the bioactive (5Z,7E)-triene in calcitriol (1). The high-performance liquid chromatography purities of batches of the synthesized calcitriol (1) were consistently more than 99.9%, with related substances listed in the USP 2023 and EP 11.0 well controlled. This robust process proved amenable to pilot scale-up and industrial production. 26,27-Hexadeutero calcitriol (4), a deuterium-labeled calcitriol derivative, is useful as the internal standard in the bioanalysis for the quantification of calcitriol in serum. 4 was efficiently synthesized in an integrated manner from hub intermediate 18 in 48% yield.
A high-performance liquid chromatography (HPLC) method has been developed for the determination of related substances in egg yolk lecithin. Chromatographic separation was achieved using a gradient ...elution on a Waters Xbridge HILIC column maintained at 35 ℃. Mobile phase A was composed of water-acetonitrile (80:20, v/v, containing 5 mM ammonium acetate), and mobile phase B was composed of acetonitrile. Analytes were monitored by a charged aerosol detector (CAD) at 50 ℃. The novel HPLC-CAD method was selective and sensitive for the determination of related substances in egg yolk lecithin in its commercial bulk batches. It was also successfully validated by the International Council for Harmonisation (ICH) guidelines. The method will be a renewal of an old Chinese Pharmacopoeia method (2020 edition). Moreover, quadrupole time-of-flight mass spectrometry (Q-TOF-MS) was integrated with HPLC to investigate phospholipid species in egg yolk lecithin. This work provides comprehensive composition profiles of egg yolk lecithin, thereby accelerating the quality control, development, and application of egg yolk lecithin.
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•Determination of related substances in egg yolk lecithin was developed and validated with HPLC-CAD.•In the profile of egg yolk lecithin, 121 phospholipid molecules were identified using HPLC-Q-TOF-MS.•This proposed method is selective, efficient, sensitive, and QC-friendly for routine analysis of related substances in egg yolk lecithin.
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•SA-FμSPE was coupled with CD-IMS for the first time.•UF sorbents were prepared through a facile strategy using low-cost chemicals.•SA-FμSPE-IMS can perform on-site screening for ...(nor-) fentanyl in urine.•The extraction procedure attains a high score in greenness evaluation.•The proposed method indicated satisfactory extraction and analysis performance.
In this work, semi-automated filter micro-solid phase extraction (SA-FμSPE) packed by urea–formaldehyde (UF) microspheres coupled with corona discharge ionization-ion mobility spectrometry (CD-IMS) was developed for the extraction and determination of fentanyl and nor-fentanyl in urine samples. UF microspheres, synthesized by aqueous polymerization, can be used to achieve effective extraction of analytes through multiple adsorption interactions such as hydrophobic, dipole–dipole and hydrogen bonding interactions. UF microspheres dispersed in methanol were packed in the filter by syringe, and the extraction process was performed by a portable automatic filter extraction device. The parameters affecting extraction efficiency (i.e., extraction time, the flow rate of extraction, pH value, amount of sorbent, ionic strength, cycle times of desorption, and type of eluent) were investigated. Then, the eluate containing fentanyl and nor-fentanyl was simultaneously analyzed by CD-IMS in positive mode. Under the optimal extraction conditions, the detection limits for the target compounds were 5.00 ng mL−1 and 8.00 ng mL−1, respectively. The spiked recoveries were in the range of 81.97 % −112.37 % by testing the real samples and validated by UHPLC-MS. Thus, the satisfactory results reveal that SA-FμSPE coupled with CD-IMS has great potential for on-site extraction and detection of fentanyl and nor-fentanyl in urine samples.
Ursodeoxycholic acid has gained increasing attention due to its recent discovery of the preventive effect on SARS-CoV-2 infection. Ursodeoxycholic acid has been included in various pharmacopoeias as ...an old drug, and the latest European Pharmacopoeia lists nine potential related substances (impurities A∼I). However, existing methods in pharmacopoeias and literature can only quantify up to five of these impurities simultaneously, and the sensitivity is inadequate, as the impurities are isomers or cholic acid analogues lacking chromophores. Herein, a novel gradient RP-HPLC method coupled to charged aerosol detection (CAD) was developed and validated for the simultaneous separation and quantification of the nine impurities in ursodeoxycholic acid. The method proved sensitive and allowed the quantification of the impurities as low as 0.02 %. Relative correction factors of the nine impurities were all within the range of 0.8–1.2 in the gradient mode by optimizing chromatographic conditions and CAD parameters. In addition, this RP-HPLC method is fully compatible with LC-MS due to the volatile additives and high percentage of the organic phase, which can be directly used for the identification of impurities. The newly developed HPLC-CAD method was successfully applied to commercial bulk drug samples, and two unknown impurities were identified by HPLC-Q-TOF-MS. The effect of CAD parameters on the linearity and correction factors was also discussed in this study. Overall, the established HPLC-CAD method can improve the methods in current pharmacopoeias and literature and contributes to understanding the impurity profile for process improvement.
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•Separation of all nine specified impurities of ursodiol by a single method.•HPLC with charged aerosol detector to detect impurities for lack of chromophores.•Method proved sensitive and correction factors of nine impurities were all 0.8–1.2.•Method is compatible with LC-MS and two unknown impurities were identified.•Overcoming the deficiency of existing methods in pharmacopoeias and literature.
Eltrombopag is an oral non-peptide thrombopoietin receptor (TPO-R) agonist indicated for the treatment of thrombocytopenia in patients with persistent or chronic immune thrombocytopenia (idiopathic ...thrombocytopenic purpura, ITP) or chronic hepatitis C infection and the treatment of severe aplastic anemia. The purpose of this research was to assess the possible impurities that may carry over to eltrombopag from its precursor Eltro-1 (3′-amino-2′-hydroxy-1,1′-biphenyl-3-carboxylic acid) and to develop a specific analytical method for the determination of these impurities. Eltro-1 samples synthesized by two different synthesis routes were investigated during the evaluation and method development studies. Besides the expected process-related impurities (Eltro-1A – Eltro‐1J), e.g., starting materials, intermediates, and/or compounds formed from their further reactions, an unknown impurity detected above 0.10% was identified by LC-MS, synthesized and fully characterized by NMR, MS and FTIR (Eltro‐1K). Accordingly, an HPLC-RP method for the determination of eleven impurities (Eltro-1A – Eltro‐1K) in Eltro-1 was developed and validated according to ICH Q2. The control limits for impurities in Eltro-1 were set at ≤ 0.15% for Eltro-1A – Eltro‐1J and ≤ 1.0% for Eltro‐1K based on fate, spike-purge and carryover studies and in accordance with the ICH M7 classification for impurities in drug substance. Eltro-1 and eleven impurities at the specification limit were separated from each other and the diluent peaks with sufficient resolution without interference. Separation was performed on a Waters XBridge C18 column (150 × 4.6 mm, 3.5 μm) at 40 °C with a 10 µL injection volume at a detection wavelength of 220 nm and 15 °C sample temperature. The gradient elution is performed at a flow rate of 1.0 mL/min for 40 min with mobile phase A (0.1% orthophosphoric acid in water) and B (acetonitrile) according to the following program: Time (min) / Acetonitrile (%): 0/0, 35/70, 36/0, 40/0. Test and standard solutions were prepared at a concentration of 1.0 mg/mL and 1.0 µg/mL, respectively, using a mixture of mobile phase A and acetonitrile (75/25) as diluent. This is the first specific, selective, sensitive, linear, precise, accurate, and robust HPLC method for the determination of Eltro-1A – Eltro‐1K in Eltro-1, which showed no significant degradation under thermal stress, photostability (UV and VIS), and standard accelerated and long-term stability conditions.
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•Evaluation of potential eltrombopag impurities from its precursor Eltro-1.•Identification (LC-MS), synthesis and characterization (NMR, MS, FTIR) of Eltro-1K.•Justification of Eltro-1K limit in Eltro-1 by fate and spike-purge study.•Development and validation (ICH Q2) of LC related substances method for Eltro-1.•The specificity of the method was also tested using Eltro-1 stressed samples.
•First enantioseparation of mepromazine enantiomers on a polysaccharide chiral column.•Validated method for the determination of chiral and achiral impurities.•Analysis of pharmacopeial reference ...substance and drug products.
A high-performance liquid chromatography method for the determination of dextromepromazine, levomepromazine sulfoxide and 2-methoxyphenothiazine in levomepromazine samples was developed. The separation of the analytes was achieved within 10 min on a stationary phase containing cellulose tris(4-methylbenzoate) as chiral selector. The mobile phase consisted of 0.1% diethylamine in methanol with a flow rate of 1.0 mL/min. The method was validated according to the International Council for Harmonization guideline Q2(R1). The detection limits based on a signal-to-noise ratio of 3 were in the range of 0.002 to 0.005 μg/mL. The method proved to be precise and accurate in the concentration range of 0.025–1.0 % for levomepromazine sulfoxide and 2-methoxyphenothiazine and 0.025% to 3.0% for dextromepromazine relative to a concentration of 0.1 mg/mL of levomepromazine, with the exception of levomepromazine sulfoxide at the 0.1% level. The method was subsequently applied to the analysis of finished pharmaceutical products as well as of reference substances of the European Pharmacopoeia.
•A novel HPLC method for determination and separation of (S)-oxiracetam and its impurities in the bulk drug of (S)-oxiracetam.•Combined with LC–MS to characterize the tentative structure of the four ...impurities of (S)-oxiracetam.•The analytical method was thoughtfully validated in terms of pharmaceutical analytical methodologies.•A reference for the quality control and stability monitor purposes of the bulk drug of (S)-oxiracetam.
(S)-oxiracetam is undergoing clinical trials as an active ingredient in the racemic oxiracetam. Here, we report a specific analytical method for analyzing (S)-oxiracetam and four related impurities in the bulk drug of (S)-oxiracetam by using high-performance liquid chromatography (HPLC) system. The chromatographic system included a Capcell pak NH2 analytical column, a mobile phase containing acetonitrile-water (95:5, v/v; pH adjusted to 2.0 with trifluoroacetic acid) at a flow rate of 1.0 mL/min, column temperature at 35 ℃ and the UV detection wavelength is set at 210 nm. This analytical method has shown effective and specific analysis for (S)-oxiracetam and four related substances. Moreover, the molecular weight and chemical structure preliminarily speculated of related substances were characterized by mass spectrometry. The methodology was verified by HPLC and results collected of the method validation included the system suitability, specificity sensitivity, linearity and accuracy, good linear correlation coefficient R2 was more than 0.9991. The analytical method developed and verified in the study, as far as we know, is the most exhaustive HPLC determination report which could be applied for the quality control and stability monitor purposes of the bulk drug of (S)-oxiracetam in the routine pharmaceutical analysis.
A new ultra‐high‐performance liquid chromatography method was developed using quality‐by‐design principles for quantifying trace‐level impurities of ibrutinib. The method utilized an ACQUITY UPLC BEH ...C18 column with a mobile phase consisting of equal parts of 0.02 M formic acid in water and 0.02 M formic acid in acetonitrile. The critical method parameters, including mobile phase pH, column temperature, and flow rate, were optimized using the design of experiments. Statistical analysis revealed the impact of these parameters on critical quality attributes. Perturbation and response surface plots illustrated the individual and interactive effects of the parameters. The optimal parameter levels were determined to be pH, 2.5; column temperature, 28°C; and flow rate, 0.55 mL/min. Confirmation experiments demonstrated the method's robustness, with the separation of impurities and unknown degradation products within a 5‐min runtime. The optimized ultra‐performance liquid chromatography method was validated according to ICH guidelines. The method exhibited linear response within the range of 0.025–100 μg/mL for ibrutinib and 0.0187–0.225 μg/mL for impurities (r2 > 0.9995), with limits of detection/limits of quantification of 0.01/0.025 and 0.015/0.0187 for ibrutinib and four impurities, respectively. Recoveries for the drug and impurities ranged from 92.69 to 102.7%, and precision was below 2% and 8% relative standard deviation for ibrutinib and impurities, respectively.
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