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Accepted Manuscript Title: Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS Authors: Yang Yang, Ying Lian, Ping Zhong, Dandan Wang, Bin Di, Bo Li PII: DOI: Reference:
S0731-7085(17)32320-8 https://doi.org/10.1016/j.jpba.2017.11.051 PBA 11625
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
14-9-2017 8-11-2017 23-11-2017
Please cite this article as: Yang Yang, Ying Lian, Ping Zhong, Dandan Wang, Bin Di, Bo Li, Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS, Journal of Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2017.11.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS Yang Yanga,1, Ying Lianb,1, Ping Zhongb, Dandan Wanga,c, Bin Dia,d*, Bo Lia,d** a
∗
authors contributed equally to this work.
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1 These
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Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China. b Henan Provincial Institute of Food and Drug Control. c Nanjing F&S Pharmatech Co. Ltd., Nanjing 211899, PR China. d Center of Drug Quality Evaluation, China Pharmaceutical University, Nanjing 210009, PR China.
Corresponding author at: Center for Drug Quality Evaluation, China Pharmaceutical University,
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24 Tongjiaxiang Road, Nanjing 210009, PR China. Phone: +86-25 8327 1269, Fax: +862583271269. E-mail addresses: [email protected] (B. Di) ∗∗
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Corresponding author at: Center for Drug Quality Evaluation, China Pharmaceutical University, 24 Tongjiaxiang Road, Nanjing 210009, PR China. Phone: +86-25 8327 1350, Fax: +862583271350. E-mail addresses: [email protected] (B. Li)
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Graphical abstract
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Highlights
Ten related substances in CoA were identified by LC-MS and seven of them were firstly reported.
A reliable HPLC method was developed and validated for quantification of related substances in CoA.
Three related substances were synthesized and confirmed with NMR.
The degradation pathway was depicted and appropriate storage condition was
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suggested.
Abstract
Ten related substances in coenzyme A (CoA) were detected using a newly developed
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gradient reverse phase high performance liquid chromatographic (HPLC) method. A highly specific and efficient LC-MS/MS method was developed to characterize process-related substances and the major degradation products, and three unknown
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related substances were further synthesized and characterized by H-NMR and
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C-NMR spectroscopy. Synthesized samples of the related substances were used for
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quantitative analysis by HPLC. The method was validated according to ICH
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guidelines with respect to specificity, precision, accuracy and linearity. The forced degradation studies included acidic, alkaline, oxidative, photolytic and thermal stress
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conditions. Furthermore, we depicted and speculated the probable mechanism of formation of related substances and the plausible fragmentation mechanism showed
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that all the related substances came from the manufacturing process. Characterization, synthesis and quantitative analysis of related substances were discussed in detail, and
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were critical for quality control, manufacturing process optimization and CoA monitoring, all of which were important to ensure the security of CoA. Key words: Coenzyme A, related substances, characterization, LC-MS/MS
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1. Introduction
Coenzyme A (CoA) is a coenzyme that is notable for its role in the synthesis and oxidation of fatty acids, as well as the oxidation of pyruvate in the citric acid cycle (TAC) [1]. The structure of coenzyme A was identified in the early 1950s [2]. The changes in the level of CoA occur at several pathological conditions, such as
diabetes, cancer and cardiac hypertrophy [3-5]. In addition, defective CoA biosynthesis is implicated in neurodegeneration with brain iron accumulation [6]. CoA is the source of the phosphopantetheine group that is assisted as a prosthetic group to proteins such as acyl carrier protein and formyltetrahydrofolate dehydrogenase [7, 8]. Due to its biological activities, CoA is applied for the treatment and prevention of metabolic imbalances by assisting in transferring fatty acids from
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the cytoplasm to mitochondria [9]. As an adjuvant therapy, is usually used in the treatment of leukopenia, idiopathic thrombocytopenic purpura and functional
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hypothermia [10, 11]. In addition, it is also useful in the therapy of chronic renal
insufficiency, acute renal insufficiency, primary cancer, lipoid nephrosis, paralytic ileus and viral hepatitis [12-17].
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CoA is applied in various treatments of diseases. It is noteworthy that drug adverse
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reaction is germane to related substances [18, 19]. The manufacturing process mainly
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relies on microbial synthesis methods; these processes are shown in Figure 1.
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Therefore, characterization, quantitative analysis and control of related substances in the bulk drug substance are an important part of regulatory assessment [20].
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A thorough literature search revealed that limited methods have been reported for the determination of CoA in related substances [21]. A few analytical methods are
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reported for the separation of known impurities, 3'-dephosphorization CoA and oxidized CoA [22, 23]. The literature search shown that the unknown related
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substances have not been studied thoroughly. A reliable method is needed for characterization and quantitative analysis of related substances in CoA. The present research described the separation of process-related substances and
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degradation products of CoA by HPLC and identified them using an LC-MS/MS method. In the study, for the identification of parent ions was used liquid chromatography - time of flight mass spectrometry (LC-TOFMS) and the characterization of fragment ions was used by liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Ten related substances (RSs) were identified, seven of these had not been previously reported. Three of these substances
(RS4, RS4 and RS8) were synthesized and confirmed by NMR as the increment contents of them increased significantly. The possible mechanisms of RSs formation were proposed, which made a great contribution to the quality promotion of CoA. The obtained data provide scientific reference for the optimization of manufacturing processes and quality assessment of CoA.
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2. Experimental 2.1 Reagents and materials
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HPLC grade methanol and acetonitrile were purchased from Tedia Company Inc. (Ohio, USA). Analytical grade ammonium acetate, ammonia, sodium hydroxide, dipotassium hydrogen phosphate and hydrochloric acid were obtained from the
Nanjing Chemical Reagent Factory (Nanjing, PR China). Water was purified through
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a PuRELAB CLASSIC system (Pall, MA, USA). CoA was supplied by Henan
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Provincial Institute of Food and Drug Control. Cysteine reference substance was
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purchased from the Chinese Food and Drug Inspection Institute (Batch No. 100329).
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5,5-dithiobis (2-nitrobenzoic acid) (Batch No. I1511022, purity≥98.0%) and
tris(2-carboxyethyl) phosphine (Batch No. F1516075, purity≥98.0%) were purchased
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from Aladdin Co. Ltd. (Shanghai, PR China). Reference related substances 3′-dephosphocoenzyme A (Batch No. SLBL2741V, purity≥90.0%), Acetyl
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coenzyme A sodium salt (Batch No. SLBC4098V, purity≥93.0%), coenzyme A disulfide (Batch No. SLBF5065V, purity≥85.0%) and adenosine 3′-phosphate (Batch
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No. SLBF9383V) were all obtained from Sigma-Aldrich Co. Ltd. (USA) 2.2 High performance liquid chromatography (HPLC) HPLC studies were performed on a Shimadzu LC-20AT HPLC system with DAD
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detector. HPLC separation was dependent on the Agilent Eclipse Plus-C18 (250 × 4.6 mm, 5 μm) system, and the mobile phase was composed of phase A (pH 7.0，20 mM ammonium acetate aqueous: methanol = 94:6) and phase B (methanol) with 1 mL/min flow rate. The gradient elution conditions were as follows: 0-7.0 min, linear from 0 to 6% B; 7.0-12.0 min, isocratic 6% B; 12.0-30.0 min linear from 6% to 25% B; and
30.0-40.0 min isocratic 0% B. The injection volume of sample was 20 μL and the column temperature was 25 ºC at 259 nm. 2.3 Liquid chromatography–tandem mass spectrometry (LC/MS/MS) HPLC conditions were used for both HPLC-UV and LC-MS detection. The parent ions for related substances studies were determined using LC-TOF mass
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spectrometric analysis and performed on an Agilent 1260 series HPLC system
combined with Agilent 6624 time-of-flight (TOF) mass spectrometry. The Agilent
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6624 time-of-flight (TOF) instrument was used with ESI source. The source
parameters were as follows: spray voltage, 4000 V; capillary temperature, 350 ºC; gas pressure, 45 psi; aux gas pressure, 10 psi. The mass spectrometer was operated in the
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full scan mode with an m/z range of 310- 1600.
Identification of fragment ions was performed using a 2010C HT series HPLC
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system (SHIMADZU, Japan), consisting of a quaternary pump solvent management
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system, an autosampler and an online degasser and Thermo TSQ Quantum mass
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spectrometry was used with an ESI source. Thermo TSQ Quantum mass spectrometry was used with an ESI source. The source parameters were as follows: spray voltage,
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4500 V; capillary temperature, 350 ºC; gas pressure, 30 psi; ion sweep gas pressure, 1.0 psi; tube lens offset, 67 V; skimmer offset, 8 V; aux gas pressure, 10 psi. The mass
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spectrometer was operated in product ion scan mode. 2.4 NMR spectroscopy
H NMR, 13C NMR and 2D NMR (COSY, HSQC, HMBC) spectra of the synthesized
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impurities were recorded on a Bruker AVANCE AV-500 spectrometer. The 1H and C chemical shift values were reported on the δ scale (ppm) relative to D2O.
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2.5 Forced degradation study For forced degradation solutions, CoA was subjected to stress conditions according to ICH guidelines [24]. The optimized stress conditions were as follows: acidic hydrolysis (2.0 M HCl, 37 ºC, 1 h), alkaline hydrolysis (2 M NaOH, 37 ºC, 1 h), oxidation (1% H2O2, 37 ºC, 1 h), thermal degradation (H2O, 60 ºC, 2 h) with water-bath heating, light degradation (natural light, 6 h), the acidic and alkaline
hydrolysis samples were neutralized before dilution, respectively. The samples were dissolved and diluted with the same dilution solvent described above to a final nominal concentration of 1 mg/mL CoA and were filtered before analysis. 3. Results and discussion 3.1 Detection of related substances by HPLC The reference related substances, 3′-dephosphocoenzyme A, acetyl coenzyme A
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sodium salt, coenzyme A disulfide and adenosine 3′-phosphate, were prepared by
dissolution in water to confirm their retention time. Using method 2.2, HPLC analysis
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revealed the presence of ten related substances at RRTs (relative retention time) 0.27, 0.34, 0.48, 0.68, 1.13, 1.23, 1.32, 2.00, 2.11 and 2.28 with respect to the principal
peak. The target related substances under study are marked RS1~10, respectively. The
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typical chromatogram highlighting the retention time of these related substances was
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shown in Figure 2.
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3.2 Mass spectrometric characteristics of Coenzyme A and its related substances CoA and related substances were characterized by LC-TOF and the mass
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spectrometric data were summarized in Table 1. According to the chromatogram,
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several peaks composed of the RS1 peak and uncertain parent ions determined by LC-MS/MS confirmed that peak1 was a mixture. The total ion chromatograms of related substances were shown in Figures 3-12. Elucidation and identification of the
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related substances were depicted as follows and the plausible fragmentation mechanism was speculated by LC-TSQ-MS/MS. In addition, we speculated the
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probable mechanism of formation of RS2~RS9, as shown in Figure 13. 3.2.1 CoA
The mass spectrum obtained for CoA showed a protonated molecular ion [M+H]+ at
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m/z 768.1246 (Figure 3A). The spectral data obtained from the MS/MS studies showed two product ion peaks at m/z 428.09 and m/z 260.68 (Figure 3B). The formation of these product ions could be explained by the dissociation mechanism shown in Figure 3C. 3.2.2 RS2 The mass spectrum of RS2 showed a protonated molecular ion at m/z 816.1095
(Figure 4A), which was identified47890 as oxidized CoA. In the MS/MS studies, RS2 gave three prominent product ion peaks at m/z 428.36, m/z 389.26 and m/z 309.18 (Figure 4B). RS2 was one of the major degradation products in oxidation stress with reduced retention under the reserve phase HPLC conditions. According to the result of HPLC and LC-MS/MS, it was reasonable to speculate that the thiol of CoA was oxidized to sulfonic group. The formation of these product ions was explained by the
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dissociation mechanism shown in Figure 4C. 3.2.3 RS3
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RS3 exhibited [M+H]+ at m/z 812.1130 and [M+2H]2+ at m/z 406.5595 in the LC-MS analysis. The MS2 analysis product ion was at m/z 428.12, which corresponded tocleavage of the phosphodiester bond and gave a fragment at m/z 305.24 (Figure 5).
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According to forced degradation, RS3 was a major process-related substance with
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increased polarity relative to CoA. It was speculated that pantothenic acid cysteine
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phosphate reacted with triphosadenine before decarboxylation during the reaction and the formation of these product ions was shown in Figure 5C.
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3.2.4 RS4
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RS4 exhibited [M+H]+ at m/z 887.1276 and [M+2H]2+ at m/z 444.0669 in LC-MS analysis (Figure 6A). RS4 was the major degradation product of both oxidation and thermal stress, with reduced retention compared to CoA. Combining the results of
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HPLC and LC-MS/MS, the RS4 was generated by the reaction between CoA and cysteine. In MS/MS studies, RS2 showed three prominent product ion peaks at m/z
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379.45 and m/z 330.01 (Figure 6B). Figure 6C presented the proposed structure and fragment pattern of RS4. 3.2.5 RS5
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The data obtained from TOF high resolution mass spectrometer showed that RS5 may possibly possess an identical formula of C21H37N7O16P3S. The protonated molecule at m/z 768.1223 fragmented to produce ions at m/z 428.04 and m/z 260.91. RS5 was the major degradation product under acidic stress, and showed a significant increase(13.5%). LC-MS/MS determination showed that the protonated molecule of RS5 was consistent with CoA, which confirmed that it was the isomeric compound of
CoA. Figure 7 presented the mass chromatogram, proposed structure and fragment pattern of RS5. 3.2.6 RS6 The protonated molecule was at m/z 843.1306 of RS6 and fragmented into product ions m/z 428.13 and m/z 336.42, which corresponded to cleavage of the phosphodiester bond. Another fragmentation pathway of the protonated molecules at
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m/z 428.13 involved cleavage of the phosphorus oxygen bond leading to ion at m/z
330.10. RS6 was the major degradation product of both oxidation and thermal stress,
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with increased retention compared to CoA. According to the result of HPLC and
LC-MS/MS, it is reasonable to speculate that to the thiol of CoA formed disulfide bond and the CoA was simultaneously added to an amino group. The mass
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chromatogram as well as the proposed structure and fragment pattern of RS5 were
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shown in Figure 8.
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3.2.7 RS7
RS7 exhibited [M+H]+ at m/z 769.1061 and [M+2H]2+ at m/z 385.0568 in LC-MS
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analysis. MS2 analysis showed product ions at m/z 508.17, which correspond to the
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cleavage of the phosphorus oxygen-bond and, produced a fragment at m/z 262.05. Another fragmentation pathway of the protonated molecules at m/z 508.17 concerned cleavage of the phosphodiester bond leading to the ion at m/z 427.93. RS7 was the
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major process-related substance with increased retention. In the light if the HPLC and LC-MS/MS data, it was reasonable to speculate that RS7 was formed by reaction of
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the product of the pantothenic acid phosphate ester and β-mercaptothion and triphosadenine during manufacturing. The mass chromatogram, proposed structure and fragment pattern of RS5 were shown in Figure 9.
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3.2.8 RS8
RS8 exhibited [M+H]+ at m/z 844.1209 and [M+H]2+ at m/z 422.5636 via MS analysis (Figure 10A). The MS2 analysis product ions was at m/z 508.17 which corresponded to cleavage of the phosphorus oxygen bond and produced fragments at m/z 336.99 and m/z 427.93 (Figure 10B). RS8 was not only a process-related substance, but also the major degradation product in acidic, alkaline, oxidation and
thermal stress with the decreased polarity. Combining the results of HPLC and LC-MS/MS, RS4 was generated by the reaction between CoA and β-mercaptothion. The fragment pattern of RS5 was shown in Figure 10C . 3.2.9 RS9 and RS10 The HPLC and LC-MS/MS results for RS9 and RS10 were consistent with coenzyme A disulfide reference and 3′-dephosphocoenzyme A reference, respectively.
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The mass chromatogram, proposed structure and fragment pattern of RS9 and RS10 were shown in Figure 11 and Figure 12, respectively.
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3.3 Synthesis and structural elucidation of RS4 3.3.1 Synthesis conditions
H2O2 (1%, 8 mL) was added to a stirred solution of coenzyme A (0.1 g) and L-Cys
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(0.1 g) dissolved in water (32 mL) and the mixture was stirred at 37 ºC for 3 h in a
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water bath. The reaction progress was monitored by HPLC (yield, 94%). The product
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was purified by preparative HPLC (LC-20A) with a Shimadzu PRC-ODS (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium
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acetate (pH 7.0) and methanol (94:6, v/v), with flow rate of 30 mL/min and UV
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detection was at 259 nm. The fractions of RS4 were lyophilized twice. HPLC detection verified the product purity (98.5%) (Figure S1). 3.3.2 Structural elucidation
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The 1H NMR spectral data for RS4 indicated that the number of hydrogen atoms agreed with the molecular formula of RS4 (Figure S2). The 13C NMR spectral data of
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RS4 showed that the line numbers were less than the carbon atoms which indicated the presence of carbon atoms with the same chemical shift. The 1H NMR and 13C NMR assignments for RS4 were summarized in table S1 and table S2. The NMR
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spectra of RS4 were shown in Figure S3. 3.4 Synthesis and structural elucidation of RS5 3.4.1 Synthesis conditions Coenzyme A (0.1 g) was dissolved in hydrochloric acid (5 mL, 1 M) and the mixture was stirred at 37 ºC for 4 h in water bath. The reaction progress was monitored by HPLC (yield, 40%). The product was purified using preparative HPLC (LC-20A)
with an Ultimate XB-C18 (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium acetate (pH 7.0) and methanol (91:9, v/v), with a flow rate of 7 mL/min and UV detection at 259 nm. The fractions of RS5 were lyophilized twice. HPLC detection verified the product purity (96.6%) (Figure S1). 3.4.2 Structural elucidation The 1H NMR spectral data of RS5 indicated that the number of hydrogen atoms fit the
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RS5 molecular formula (Figure S2). The 13C NMR spectral data of RS5 showed that
line numbers were less than the carbon atoms which indicated the presence of carbon
were summarized in Table S3 and Table S4.
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atoms with same chemical shift. The 1H NMR and 13C NMR assignments for RS5
The NMR spectra of RS5 were shown
in Figure S4.
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3.5 Synthesis and structural elucidation of RS8
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3.5.1 Synthesis conditions
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H2O2 (1%, 2 mL) and β-mercaptoethanol (50 μL) were added to a stirring solution of coenzyme A (0.1 g) dissolved in buffer (pH=8, 32 mL) and the mixture was stirred at
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37 ºC for 20 min in a water bath. The progress of the reaction was monitored by HPLC (yield, 95%). The product was purified by preparative HPLC (LC-20A) with a
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Shimadzu PRC-ODS (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium acetate (pH 7.0) and methanol (91:9, v/v), with flow rate of
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30 mL/min and UV detection was at 259 nm. The fractions of RS8 were lyophilized twice. HPLC detection verified the product purity (98.5%) (Figure S1).
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3.5.2 Structural elucidation The 1H NMR spectral data for RS8 indicated that the number of hydrogen atoms agreed with the RS8 molecular formula (Figure S2). The 13C NMR spectral data of
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RS8 showed that line numbers were less than the carbon atoms which indicated thepresence of carbon atoms with the same chemical shift. The 1H NMR and 13C NMR assignments for RS4 were summarized in Table S5 and Table S6. The NMR spectra of RS8 were shown in Figure S5. 3.6 Optimization of HPLC method Initially, different types of HPLC columns, such as Agilent C8 (250 × 4.6 mm,5μm)
column, Agilent Eclipse Plus C18 (250 × 4.6 mm, 5μm) column, Agilent ZORBAX HILIC PLUS column (4.6 mm×100 mm, 3.5 μm) were tested to analysis CoA. The performance characteristics of the columns were mainly assessed for their ability to separate CoA and the related substances. The best resolution for the critical peaks was obtained on the Discovery Agilent Eclipse Plus C18 column which was thereafter used for further optimization of the method.
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Different gradient profile and pH values were explored all together to develop a
selective separation method. The retention of CoA was enhanced as pH decreased in
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the tested pH from 5.0-8.0 and the parameters and chromatogram were shown in
Figure S7 and Table S7. Shape symmetrical peaks with good resolution between CoA and its RSs were achieved at pH 7.0. The mechanism of CoA retention behavior vs.
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pH was proposed. The non-ionic proportion of CoA under acidic conditions was
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greater than that under mild alkaline and neutral conditions due to the presence of the
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adenine group in CoA. However, under acidic conditions, tailing peak appeared that was attributed to the ionic species of CoA that were simultaneously present in ionic
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and non-ionic forms. Accordingly, the acid and alkaline conditions were not suitable
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for CoA analysis.
Different conditions of mobile phase were tested. The method has been optimized by comparing separation of related substance, shape symmetrical peaks of CoA and the
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number of related substances. Two gradient modes were developed with different initial proportions of mobile phase which named method-A (phase A: methanol/
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ammonium acetate aqueous =90/10, phase B: methanol) and method-B (phase A: methanol/ ammonium acetate aqueous=94/6, phase B: methanol). The gradient elution conditions were as follows: 0-30.0 min, linear from 0 to 25% B; 30.0-40.0 min
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isocratic 0% B. The polar impurities cannot be well separated with method-A. However, a distorted peak of CoA was observed when decreased the initial proportion to 6% of organic phase (method-B). The separation of related substances was not satisfactory by a continuous gradient elution program. For the separation of RS5, RS6 and RS7 as well as the shape symmetrical peaks, the gradient profile was optimized. An isocratic hold process was applied in the method B to simultaneously improve the
separation and resolve the problem of distorted peak of CoA. Finally, the HPLC conditions were optimized by studying the effect of column types, buffer pH and gradient modes which was shown in section 2.2. 3.7 Validation of HPLC method 3.7.1 Quantitative method
concentration of 1 mg/mL for the analysis of related substances.
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Test solutions were accurately prepared by dissolving CoA in water to a final
Normally, three quantitative methods, area normalization method, main component
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self-compare method without a correction factor and main component self-compare method with a correction factor, were used to detect related substances. Correction factor is essential, given the difference responses of related substances and the
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principal component. In this experiment, three methods were applied to detect RS4,
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RS5, RS8 and RS10 and the result was shown in Table 2. The result showed that the
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appropriate method was main component self-compare method. Therefore, the RS4, RS5, RS8 and RS10 were detected using the main component self-compare method
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with correction factor and the other RSs were detected by main component
3.7.2 Specificity
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self-compare method without a correction factor.
The specificity of the method was checked using test solutions and the forced
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degradation solutions described in Section 2.5 using a DAD detector to determine the spectral peak purity of all of the chromatographic peaks. No interfering coeluting
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peaks were observed in the blank solutions, and the resolution between adjacent peaks was above 3.0 demonstrating adequate selectivity of the HPLC method (Figure 14). Assay studies were performed for stress samples against the CoA qualified reference
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standards. The mass balance results were calculated for all of the stressed samples and found to be 99.3–101.3% at 259 nm, as shown in Table 3. 3.7.3 Limit of detection (LOD) and limit of quantitation (LOQ) LOD and LOQ were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively. The RSD of the areas for six replicate injections at the LOQ concentration were found to be below 10%, and the signal-to-noise ratios at LOD and LOQ concentration were
found to be below 3 and 10, respectively. The determined LOD values for RS4, RS5, RS8 and RS10 were 0.206, 0.084, 0.102 and 0.060 μg/mL and LOQ values were 0.514, 0.169, 0.203 and 0.124 μg/mL, respectively. The unknown impurities were represented by CoA and the LOD/LOQ was 0.060 and 0.18 μg/mL, respectively. 3.7.4 Linearity Linearity of the detector response was examined for the assay and related substances.
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For the CoA method, eight concentrations (0.45 μg/mL, 0.90 μg/mL, 1.8 μg/mL, 4.5 μg/mL, 9.0 μg/mL, 18 μg/mL, 45 μg/mL and 90 μg/mL) were prepared. For the
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related substances test RS4, RS5, RS8 and RS10 were examined by preparing
standard solution at eight different levels ranging from 0.5 μg/mL to 103 μg/mL, 0.42 μg/mL to 84 μg/mL, 0.51 μg/mL to 102 μg/mL, and 0.50 μg/mL to 100 μg/mL,
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respectively. The calibration equation, correlation coefficient (r) and correction factor
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of CoA and related substances were shown in Table 4 and the regression curves were
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shown in Figure S6. 3.7.5 Accuracy
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Accuracy of the method was determined by spiking known impurities at 80%, 100%
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and 120% w/w of their specified limits in dosage form. The recovery of RS4, RS5, RS8 and RS10 ranged from 98.40%~103.48% (w/w) for the assay method and the relative standard deviation was less than 2%. The percentage recoveries were
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recorded in Table 4.
3.7.6 Precision and repeatability
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The reference substance CoA solution (10 μg/mL) was serially injected six times. The relative standard deviation of peak areas was 0.2%, showing good precision. Prepared six CoA solutions and their diluted solutions were injected to evaluate the
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repeatability which was shown in Table 5. According to result, the RSD of content was less than 2%, indicating good repeatability for the method. 3.7.7 Stability of CoA solution Solution stability was tested by leaving spiked sample solutions in tightly capped volumetric flasks at 4 ℃ for 12 h. No significant change in peak areas was observed during the solution stability experiments. The results confirmed that the
solutions were stable for up to 12 h during the determination. The CoA solution (1 mg/mL) was tested after 24 h at 4 ℃. The RS3, RS4 and RS9 were unstable and the result of stability was shown in Table 6. Therefore, samples have to be prepared freshly before use. 3.7.8 Robustness To determine the robustness of the method, experimental conditions were deliberately
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changed: pH (7.0 ± 0.5), flow rate (1.0 ± 0.2 mL/min), detection wavelength (259 ± 5 nm), column temperature (25 ± 5 ℃) and different instruments (Agilent 1260 and
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Shimadzu 2010). Furthermore, mobile phase A was adjusted to 4% MET, 6% MET
(nominal) and 8% MET. The results obtained were assessed for system suitability to ensure that the separation requirements were maintained under the altered conditions
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and robustness was assessed by examination the content of related substances.
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Cumulative RSD was calculated for the RS5, RS8 and RS10 in the standard solution
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and was found to be less than 10.0%. Results were summarized in Table 7. 3.8 Application of the method: analysis of commercial samples
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The proposed LC method was applied for the determination of related substances
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determination of CoA bulk drug (Sample A~I) available on the market. The results were summarized in Table 8. Among them, RS5, RS7, RS8, RS9 and RS10 were present at greater than 1.0%.
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4. Conclusion
A complex component of CoA was due to the microbial synthesis and the related
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substances of the highest contents in CoA were characterized. The main related substances in CoA were characterized by LC-MS/MS and product ions were predicted. Among them, RS4, RS5, and RS8 were further synthesized and elucidated by NMR
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spectroscopy. In addition, a reliable HPLC method was developed and validated for quantitation analysis of related substances. The probable mechanism of formation of related substances was speculated. All the related substances came from the manufacturing process and some could be produced under the forced degradation conditions. Among them, RS2/6 were the major degradation products under oxidation stress, RS4/8/9 were the major degradation products under thermal degradation, RS5
was the major degradation product under acidic hydrolysis and RS8 was the major degradation product under alkaline hydrolysis. Related substance research is very important to the security, the quality promotion and assessment of drugs. The result reminds us to control the specific steps purposefully, as they are valuable for quality
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control in the manufacturing of CoA.
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Acknowledgment
This work was financially supported by Key Laboratory on Protein Chemistry and Structural Biology (No. 2016ZPT005), Supported by the Fundamental Research
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Appendix A. supplementary data
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The authors have declared no conflict of interest.
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Funds for the Central Universities (No. 2632017ZD07).
Supplementary data associated with this article described the NMR spectrograms and
References
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the MS spectrograms
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[1] F.P. Miller, A.F. Vandome, J. Mcbrewster, Coenzyme A, Alphascript Publishing 2010. [2] W.H.G. Devries, J.S. W. M. Evans, J.D. Gregory, G.D. Novelli, M. Soodak, F. Lipmann, Purification of coenzyme a from fermentation sources and its further partial identification, J. Am. Chem. Soc. 72 (1950) 4838-4838. [3] R.J. Perry, J.P.G. Camporez, R. Kursawe, P.M. Titchenell, D. Zhang, C.J. Perry, M.J. Jurczak, A. Abudukadier, M.S. Han, X.M. Zhang, H.B. Ruan, X. Yang, S. Caprio, S.M. Kaech, H.S. Sul, M.J. Birnbaum, R.J. Davis, G.W. Cline, K.F. Petersen, G.I. Shulman, Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes, Cell. 160 (2015) 745-758. [4] J.J. Kamphorst, M.K. Chung, J. Fan, J.D. Rabinowitz, Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate, Cancer. Metab. 2 (2014) 23-30. [5] L.G. Abo Alrob O, Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose
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oxidation. Biochem Soc Trans, Biochem. Soc. Trans. 42 (2014) 1043-1051. [6] A.M. Colombelli C, Tiranti V, Defective lipid metabolism in neurodegeneration with brain iron accumulation (NBIA) syndromes: not only a matter of iron, J. Inherited Metab. Dis. 38 (2015) 123-136. [7] T. Kupke, P. Hernándezacosta, F.A. Culiáñezmacià, 4'-phosphopantetheine and coenzyme A biosynthesis in plants, J. Biol. Chem. 278 (2003) 38229-38237. [8] J. Grünewald, Y. Jin, J. Vance, J. Read, X. Wang, Y. Wan, H. Zhou, W. Ou, H.E. Klock, E.C. Peters, Optimization of an Enzymatic Antibody-Drug Conjugation Approach Based on Coenzyme A Analogs, Bioconjugate Chem. 28 (2017) 1906-1915. [9] J.G. Nijssen, H.V.D. Bosch, Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes, Biochim. Biophys. Acta. 875 (1986) 458-464. [10] L. Jun, Clinical Application of Leucogen Combined with Coenzyme A in Treatment of Leukopenia, Chinese Journal of General Practice. 12 (2012) 1880-1911. [11] M. Takamiya, K. Saigusa, K. Dewa, DNA Microarray Analysis of the Mouse Adrenal Gland for the Detection of Hypothermia Biomarkers: Potential Usefulness for Forensic Investigation, Ther. Hypothermia. Tem. 3 (2013) 63-73. [12] M. G, Clinical trial of coenzyme A in hepatopathies, Minerva. Med. 67 (1976) 512-518. [13] M. Derot, M. Rathery, Coenzyme A in the treatment of chronic renal insuficiency, Therapie. 15 (1960) 603-611. [14] M. Derot, M. Rathery, The place of coenzyme A in the treatment of acute anuric renal insufficiency, Therapie. 15 (1960) 591-602. [15] M. Payet, M. Sankale, R. Camain, Y. Le Duc, Trial treatment of primary cancer of the liver by coenzyme A, Therapie. 15 (1960) 143-147. [16] M. Payet, M. Sankale, P. Pene, Y. Leduc, M. Moulanier, The treatment of lipoid nephrosis by coenzyme A, Therapie. 15 (1960) 217-230. [17] C. Amato, A. Majani, Paralytic ileus caused by pseudourological acute abdomen. Treatment with coenzyme A, Minerva chirurgica. 18 (1963) 683-686. [18] K. J, Impurity linked to adverse events, Chem. Eng. News. 86 (2008) 32-34. [19] W.B. Cong Luoluo, Guo Hongzhu, Shao Mingli, Data mining and association analysis of total related substance and adverse reaction rate of antibiotics, Zhongguo Xinyao Zazhi. 25 (2016) 964-967. [20] ICH guideline: Impurities in New Drug Substances Q3A (R2), 2006. [21] C. Fengyun, Determination of related substances in Coenzyme A with HPLC, Clin. Med. 25 (2005) 35-36. [22] L. Fuxiong, Determination of related substances in Coenzyme A for injection by gradient elution, Zhongguo Shenghua Yaowu Zazhi. 30 (2009) 124-126. [23] L. Ying, Study on the Quality Control for Coenzyme A, Pharmaceutical Sciences, Zhengzhou University, 2009. [24] ICH guidelines: Q1A (R2): Stability Testing of New Drug Substances and Products 2003.
Figure legends
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Figure 1. The synthesis processes of CoA. Figure 2. Chromatogram of Coenzyme A and ten related substances. Figure 3. The mass chromatogram and plausible scheme for fragmentations of CoA. Figure 4. The mass chromatogram and plausible scheme for fragmentations of RS2. Figure 5. The mass chromatogram and plausible scheme for fragmentations of RS3. Figure 6. The mass chromatogram and plausible scheme for fragmentations of RS4. Figure 7. The mass chromatogram and plausible scheme for fragmentations of RS5. Figure 8. The mass chromatogram and plausible scheme for fragmentations of RS6. Figure 9. The mass chromatogram and plausible scheme for fragmentations of RS7. Figure 10. The mass chromatogram and plausible scheme for fragmentations of RS8. Figure 11. The mass chromatogram and plausible scheme for fragmentations of RS9. Figure 12. The mass chromatogram and plausible scheme for fragmentations of RS10. Figure 13. Probable mechanism of formation of RS2~RS10. Figure 14. Typical HPLC chromatograms of CoA and its degradation products formed under (A) 2.0 M HCl, 37 ºC, 1 h, (B) 2 M NaOH, 37 ºC, 1 h, (C) 1% H2O2, 37 ºC, 1 h, (D) H2O, 60 ºC, 2 h, (E) natural light, 6 h.
A ED
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A
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N
A
M
A ED
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SC R
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N
A
M
A ED
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U
N
A
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N
A
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A
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Table 1 TOF mass spectra results of CoA and its related substances. Theoretical
Proposed
Observed ion related
tR (min)
Error ion mass
Molecular
Product ions
(m/z)
Formula [M+H]
768.1225
C21H37N7O16P3S
---
---
mass (m/z) substances CoA
8.81
768.1246
(ppm)
RS1
2.38
353.0834,
2.73
428.09, 260.68
SC R
337.0029,
+
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Name of
---
3.29
816.1095
816.1072
C21H37N7O19P3S
2.82
428.36, 389.26, 309.18
RS3
4.60
812.1130
812.1123
C22H37N7O18P3S
0.86
428.12, 305.24
RS4
6.47
887.1276
887.1266
C24H42N8O18P3S2
1.13
379.45, 330.01
RS5
10.30
768.1224
768.1225
C21H37N7O16P3S
-0.13
428.04, 260.91
RS6
11.02
843.1360
843.1368
C23H42N8O16P3S2
-0.95
428.13, 336.42, 330.10
RS7
11.91
769.1061
769.1065
C21H36N6O17P3S
-0.52
508.17, 427.93, 262.05
RS8
17.85
844.1209
844.1208
C23H41N7O17P3S2
0.12
508.17, 427.93. 336.99
767.1146
[C42H72N14O32P6S2]2+
0.00
617.92, 519.99, 428.58
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RS10
19.52
20.34
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RS9
767.1146
688.1558
A
RS2
N
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403.0299
---
513.64, 428.22, 688.1562
C21H36N7O13P2S
-0.58 348.48, 261.17
Table 2 Compare the results of RS contents by different quantitative methods
Content o Content
Content o Content o um Co
Total cont
f CoA
f
ent of RS
f
ntent o
RS5（%） RS8（%） RS10（%） f RS
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（%）
of
4.3
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Quantitative method
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Maxim
3.51
4.5
0.65
3.06
（%）
（%）
7
Main-component self-co ction factor
0.14 4
Main-component self-co mpare method with correcti
4 0.17
5
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5 4.55
2
3.34
7
0.53
1
5.65
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on factor
1
0.50
A
mpare method without corre
4.33
M
method
0.13
N
Area normalization
0
31
9
5
15.30 3
54
16.16 9
5.6 52
16.96 3
Table 3 The parameters of forced degradation studies.
Resolution between CoA Cont
Samples
ent（%）
CoA peak and nearest p
Mass bala nce （%）
Peak puri ty of CoA
ysis Oxidation degr adation Thermal degrad ation Light degradati
3.2
100.5
48.195
3.7
72.181
4.0
85.454
3.3
85.312
3.9
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M
on
68.580
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Alkaline hydrol
——
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sis
4.0
N
Acidic hydroly
85.018
A
Sample
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eak 0.979 0.948
100.8
0.990
101.3
0.986
99.3
0.967
99.4
0.984
Table 4 Linearity and recovery data.
Analyte
Calibration
Correlation
Correction
equation
coefficient (r)
factor
Recovery (%)
recovery
1
---
RS 4
y=15426x-8852.1
0.9999
1.22
RS 5
1
1.24
1
RS 10
y=21551x-6824.8
1
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*the correction factor is slope CoA/slope RS
A
99.637
0.812
99.813
0.903
102.791
0.565
1.22
A
y=15324x-5660.0
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RS 8
1.164
N
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y=15120x-4885.5
101.828
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y=18766x-4408.8
---
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--CoA
RSD of
0.87
Table 5 The repeatability of related substances.
RSD (%, n=6)
RS2
0.598
1.1
RS3
0.621
0.7
RS4
0.076
1.5
RS5
5.152
0.2
RS6
0.260
0.1
RS7
1.977
0
RS8
0.616
RS9
0.833
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Oher RS
2.293
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N 0.1
A
M 2.947
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RS10
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Content（%） Average
1.2 0.1 0.6
Table 6 Stability data. Content of CoA
Content of RS3
Content of RS4
Content of RS9
（%）
（%）
（%）
（%）
Room
Room
Room
Room
（h）
re
4
84.825
0.816
84.91
0.86 0.858
0.10
0.122
5
84.959
85.06
0.88
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8
M
0.88
84.96
3 0.66
0.685 9
1
0.11
0.66
0.139
0.731 1
4
0.90
0.11
0.66
0.168
0.774
3
1
3
7
85.00
0.92
0.11
0.67
0.918
0.184
0.818
9
5
7
84.93
0.95
0.12
84.564
A
24
0.65
5
0.892
84.774
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16
84.82
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12
4℃
0.653
7
A
8
temperatu
0.10
0.107 6
4℃
re
0.81
5
84.916
temperatu re
84.82 0
4℃
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re
temperatu
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4℃
N
temperatu
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Time
0.924 4
0.196 6
8
0.896
0.69
11.8
2
8
RSD 0.2 （%）
0.1
4.6
5.5
23.3
6.7
Table 7 The data of robustness RSD of concentration (%) RS10
A
2.43
2.67
3.89
B
1.86
0.48
2.10
C
2.68
3.82
1.81
D
1.09
7.35
0.35
E
1.37
0.86
1.73
F
1.01
1.24
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RS8
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RS5
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Conditions
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0.83
A
*A: pH (7.0 ± 0.5); B: initial proportion of phase A (4% MET, 6% MET and 8% MET); C:
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instruments (Agilent 1260 and Shimadzu 2010); D: column temperature (25 ± 5 ℃); E:
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flow rate (1.0 ± 0.2 mL/min); F: detection wavelength (259 ± 5 nm).
Table 8 Results of impurity determination for CoA drug substance samples (A-I).
Sample
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Content (%) Other
RS3
RS4
RS5
RS6
RS7
RS8
RS9
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RS2
RS10
RSs
0.202
0.675
0.097
3.751
c
1.572
0.737
1.088
2.911
1.063
Sample B
0.084
0.579
0.11
5.293
0.197
2.381
0.71
0.605
2.574
1.308
Sample C
0.074
0.612
0.126
3.875
0.165
Sample D
0.164
0.665
0.16
4.126
Sample E
0.169
0.857
0.076
Sample F
0.546
0.889
0.175
0.478
0.857
0.58
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Sample H
0.759
0.556
2.338
0.772
0.176
1.447
0.943
0.582
4.041
1.028
3.922
0.151
2.43
1.979
0.567
6.626
1.863
5.652
0.258
2.058
0.65
0.863
3.065
2.806
M
A
N
1.492
ED
G
0.168
5.304
0.246
1.938
0.605
0.852
2.9
2.847
0.87
0.179
5.379
0.253
1.969
0.627
0.819
2.934
2.698
0.867
0.195
5.205
0.16
1.063
2.47
1.34
1.456
1.56
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Sample
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Sample A
Sam
0.375
A
ple I
S0731-7085(17)32320-8 https://doi.org/10.1016/j.jpba.2017.11.051 PBA 11625
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
14-9-2017 8-11-2017 23-11-2017
Please cite this article as: Yang Yang, Ying Lian, Ping Zhong, Dandan Wang, Bin Di, Bo Li, Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS, Journal of Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2017.11.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization and quantitative analysis of related substances in Coenzyme A by HPLC and LC-MS/MS Yang Yanga,1, Ying Lianb,1, Ping Zhongb, Dandan Wanga,c, Bin Dia,d*, Bo Lia,d** a
∗
authors contributed equally to this work.
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1 These
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Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China. b Henan Provincial Institute of Food and Drug Control. c Nanjing F&S Pharmatech Co. Ltd., Nanjing 211899, PR China. d Center of Drug Quality Evaluation, China Pharmaceutical University, Nanjing 210009, PR China.
Corresponding author at: Center for Drug Quality Evaluation, China Pharmaceutical University,
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24 Tongjiaxiang Road, Nanjing 210009, PR China. Phone: +86-25 8327 1269, Fax: +862583271269. E-mail addresses: [email protected] (B. Di) ∗∗
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Corresponding author at: Center for Drug Quality Evaluation, China Pharmaceutical University, 24 Tongjiaxiang Road, Nanjing 210009, PR China. Phone: +86-25 8327 1350, Fax: +862583271350. E-mail addresses: [email protected] (B. Li)
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Graphical abstract
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Highlights
Ten related substances in CoA were identified by LC-MS and seven of them were firstly reported.
A reliable HPLC method was developed and validated for quantification of related substances in CoA.
Three related substances were synthesized and confirmed with NMR.
The degradation pathway was depicted and appropriate storage condition was
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suggested.
Abstract
Ten related substances in coenzyme A (CoA) were detected using a newly developed
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gradient reverse phase high performance liquid chromatographic (HPLC) method. A highly specific and efficient LC-MS/MS method was developed to characterize process-related substances and the major degradation products, and three unknown
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related substances were further synthesized and characterized by H-NMR and
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C-NMR spectroscopy. Synthesized samples of the related substances were used for
A
quantitative analysis by HPLC. The method was validated according to ICH
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guidelines with respect to specificity, precision, accuracy and linearity. The forced degradation studies included acidic, alkaline, oxidative, photolytic and thermal stress
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conditions. Furthermore, we depicted and speculated the probable mechanism of formation of related substances and the plausible fragmentation mechanism showed
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that all the related substances came from the manufacturing process. Characterization, synthesis and quantitative analysis of related substances were discussed in detail, and
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were critical for quality control, manufacturing process optimization and CoA monitoring, all of which were important to ensure the security of CoA. Key words: Coenzyme A, related substances, characterization, LC-MS/MS
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1. Introduction
Coenzyme A (CoA) is a coenzyme that is notable for its role in the synthesis and oxidation of fatty acids, as well as the oxidation of pyruvate in the citric acid cycle (TAC) [1]. The structure of coenzyme A was identified in the early 1950s [2]. The changes in the level of CoA occur at several pathological conditions, such as
diabetes, cancer and cardiac hypertrophy [3-5]. In addition, defective CoA biosynthesis is implicated in neurodegeneration with brain iron accumulation [6]. CoA is the source of the phosphopantetheine group that is assisted as a prosthetic group to proteins such as acyl carrier protein and formyltetrahydrofolate dehydrogenase [7, 8]. Due to its biological activities, CoA is applied for the treatment and prevention of metabolic imbalances by assisting in transferring fatty acids from
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the cytoplasm to mitochondria [9]. As an adjuvant therapy, is usually used in the treatment of leukopenia, idiopathic thrombocytopenic purpura and functional
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hypothermia [10, 11]. In addition, it is also useful in the therapy of chronic renal
insufficiency, acute renal insufficiency, primary cancer, lipoid nephrosis, paralytic ileus and viral hepatitis [12-17].
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CoA is applied in various treatments of diseases. It is noteworthy that drug adverse
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reaction is germane to related substances [18, 19]. The manufacturing process mainly
A
relies on microbial synthesis methods; these processes are shown in Figure 1.
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Therefore, characterization, quantitative analysis and control of related substances in the bulk drug substance are an important part of regulatory assessment [20].
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A thorough literature search revealed that limited methods have been reported for the determination of CoA in related substances [21]. A few analytical methods are
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reported for the separation of known impurities, 3'-dephosphorization CoA and oxidized CoA [22, 23]. The literature search shown that the unknown related
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substances have not been studied thoroughly. A reliable method is needed for characterization and quantitative analysis of related substances in CoA. The present research described the separation of process-related substances and
A
degradation products of CoA by HPLC and identified them using an LC-MS/MS method. In the study, for the identification of parent ions was used liquid chromatography - time of flight mass spectrometry (LC-TOFMS) and the characterization of fragment ions was used by liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Ten related substances (RSs) were identified, seven of these had not been previously reported. Three of these substances
(RS4, RS4 and RS8) were synthesized and confirmed by NMR as the increment contents of them increased significantly. The possible mechanisms of RSs formation were proposed, which made a great contribution to the quality promotion of CoA. The obtained data provide scientific reference for the optimization of manufacturing processes and quality assessment of CoA.
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2. Experimental 2.1 Reagents and materials
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HPLC grade methanol and acetonitrile were purchased from Tedia Company Inc. (Ohio, USA). Analytical grade ammonium acetate, ammonia, sodium hydroxide, dipotassium hydrogen phosphate and hydrochloric acid were obtained from the
Nanjing Chemical Reagent Factory (Nanjing, PR China). Water was purified through
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a PuRELAB CLASSIC system (Pall, MA, USA). CoA was supplied by Henan
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Provincial Institute of Food and Drug Control. Cysteine reference substance was
A
purchased from the Chinese Food and Drug Inspection Institute (Batch No. 100329).
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5,5-dithiobis (2-nitrobenzoic acid) (Batch No. I1511022, purity≥98.0%) and
tris(2-carboxyethyl) phosphine (Batch No. F1516075, purity≥98.0%) were purchased
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from Aladdin Co. Ltd. (Shanghai, PR China). Reference related substances 3′-dephosphocoenzyme A (Batch No. SLBL2741V, purity≥90.0%), Acetyl
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coenzyme A sodium salt (Batch No. SLBC4098V, purity≥93.0%), coenzyme A disulfide (Batch No. SLBF5065V, purity≥85.0%) and adenosine 3′-phosphate (Batch
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No. SLBF9383V) were all obtained from Sigma-Aldrich Co. Ltd. (USA) 2.2 High performance liquid chromatography (HPLC) HPLC studies were performed on a Shimadzu LC-20AT HPLC system with DAD
A
detector. HPLC separation was dependent on the Agilent Eclipse Plus-C18 (250 × 4.6 mm, 5 μm) system, and the mobile phase was composed of phase A (pH 7.0，20 mM ammonium acetate aqueous: methanol = 94:6) and phase B (methanol) with 1 mL/min flow rate. The gradient elution conditions were as follows: 0-7.0 min, linear from 0 to 6% B; 7.0-12.0 min, isocratic 6% B; 12.0-30.0 min linear from 6% to 25% B; and
30.0-40.0 min isocratic 0% B. The injection volume of sample was 20 μL and the column temperature was 25 ºC at 259 nm. 2.3 Liquid chromatography–tandem mass spectrometry (LC/MS/MS) HPLC conditions were used for both HPLC-UV and LC-MS detection. The parent ions for related substances studies were determined using LC-TOF mass
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spectrometric analysis and performed on an Agilent 1260 series HPLC system
combined with Agilent 6624 time-of-flight (TOF) mass spectrometry. The Agilent
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6624 time-of-flight (TOF) instrument was used with ESI source. The source
parameters were as follows: spray voltage, 4000 V; capillary temperature, 350 ºC; gas pressure, 45 psi; aux gas pressure, 10 psi. The mass spectrometer was operated in the
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full scan mode with an m/z range of 310- 1600.
Identification of fragment ions was performed using a 2010C HT series HPLC
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system (SHIMADZU, Japan), consisting of a quaternary pump solvent management
A
system, an autosampler and an online degasser and Thermo TSQ Quantum mass
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spectrometry was used with an ESI source. Thermo TSQ Quantum mass spectrometry was used with an ESI source. The source parameters were as follows: spray voltage,
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4500 V; capillary temperature, 350 ºC; gas pressure, 30 psi; ion sweep gas pressure, 1.0 psi; tube lens offset, 67 V; skimmer offset, 8 V; aux gas pressure, 10 psi. The mass
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spectrometer was operated in product ion scan mode. 2.4 NMR spectroscopy
H NMR, 13C NMR and 2D NMR (COSY, HSQC, HMBC) spectra of the synthesized
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1
impurities were recorded on a Bruker AVANCE AV-500 spectrometer. The 1H and C chemical shift values were reported on the δ scale (ppm) relative to D2O.
13
A
2.5 Forced degradation study For forced degradation solutions, CoA was subjected to stress conditions according to ICH guidelines [24]. The optimized stress conditions were as follows: acidic hydrolysis (2.0 M HCl, 37 ºC, 1 h), alkaline hydrolysis (2 M NaOH, 37 ºC, 1 h), oxidation (1% H2O2, 37 ºC, 1 h), thermal degradation (H2O, 60 ºC, 2 h) with water-bath heating, light degradation (natural light, 6 h), the acidic and alkaline
hydrolysis samples were neutralized before dilution, respectively. The samples were dissolved and diluted with the same dilution solvent described above to a final nominal concentration of 1 mg/mL CoA and were filtered before analysis. 3. Results and discussion 3.1 Detection of related substances by HPLC The reference related substances, 3′-dephosphocoenzyme A, acetyl coenzyme A
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sodium salt, coenzyme A disulfide and adenosine 3′-phosphate, were prepared by
dissolution in water to confirm their retention time. Using method 2.2, HPLC analysis
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revealed the presence of ten related substances at RRTs (relative retention time) 0.27, 0.34, 0.48, 0.68, 1.13, 1.23, 1.32, 2.00, 2.11 and 2.28 with respect to the principal
peak. The target related substances under study are marked RS1~10, respectively. The
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typical chromatogram highlighting the retention time of these related substances was
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shown in Figure 2.
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3.2 Mass spectrometric characteristics of Coenzyme A and its related substances CoA and related substances were characterized by LC-TOF and the mass
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spectrometric data were summarized in Table 1. According to the chromatogram,
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several peaks composed of the RS1 peak and uncertain parent ions determined by LC-MS/MS confirmed that peak1 was a mixture. The total ion chromatograms of related substances were shown in Figures 3-12. Elucidation and identification of the
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related substances were depicted as follows and the plausible fragmentation mechanism was speculated by LC-TSQ-MS/MS. In addition, we speculated the
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probable mechanism of formation of RS2~RS9, as shown in Figure 13. 3.2.1 CoA
The mass spectrum obtained for CoA showed a protonated molecular ion [M+H]+ at
A
m/z 768.1246 (Figure 3A). The spectral data obtained from the MS/MS studies showed two product ion peaks at m/z 428.09 and m/z 260.68 (Figure 3B). The formation of these product ions could be explained by the dissociation mechanism shown in Figure 3C. 3.2.2 RS2 The mass spectrum of RS2 showed a protonated molecular ion at m/z 816.1095
(Figure 4A), which was identified47890 as oxidized CoA. In the MS/MS studies, RS2 gave three prominent product ion peaks at m/z 428.36, m/z 389.26 and m/z 309.18 (Figure 4B). RS2 was one of the major degradation products in oxidation stress with reduced retention under the reserve phase HPLC conditions. According to the result of HPLC and LC-MS/MS, it was reasonable to speculate that the thiol of CoA was oxidized to sulfonic group. The formation of these product ions was explained by the
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dissociation mechanism shown in Figure 4C. 3.2.3 RS3
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RS3 exhibited [M+H]+ at m/z 812.1130 and [M+2H]2+ at m/z 406.5595 in the LC-MS analysis. The MS2 analysis product ion was at m/z 428.12, which corresponded tocleavage of the phosphodiester bond and gave a fragment at m/z 305.24 (Figure 5).
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According to forced degradation, RS3 was a major process-related substance with
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increased polarity relative to CoA. It was speculated that pantothenic acid cysteine
A
phosphate reacted with triphosadenine before decarboxylation during the reaction and the formation of these product ions was shown in Figure 5C.
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3.2.4 RS4
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RS4 exhibited [M+H]+ at m/z 887.1276 and [M+2H]2+ at m/z 444.0669 in LC-MS analysis (Figure 6A). RS4 was the major degradation product of both oxidation and thermal stress, with reduced retention compared to CoA. Combining the results of
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HPLC and LC-MS/MS, the RS4 was generated by the reaction between CoA and cysteine. In MS/MS studies, RS2 showed three prominent product ion peaks at m/z
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379.45 and m/z 330.01 (Figure 6B). Figure 6C presented the proposed structure and fragment pattern of RS4. 3.2.5 RS5
A
The data obtained from TOF high resolution mass spectrometer showed that RS5 may possibly possess an identical formula of C21H37N7O16P3S. The protonated molecule at m/z 768.1223 fragmented to produce ions at m/z 428.04 and m/z 260.91. RS5 was the major degradation product under acidic stress, and showed a significant increase(13.5%). LC-MS/MS determination showed that the protonated molecule of RS5 was consistent with CoA, which confirmed that it was the isomeric compound of
CoA. Figure 7 presented the mass chromatogram, proposed structure and fragment pattern of RS5. 3.2.6 RS6 The protonated molecule was at m/z 843.1306 of RS6 and fragmented into product ions m/z 428.13 and m/z 336.42, which corresponded to cleavage of the phosphodiester bond. Another fragmentation pathway of the protonated molecules at
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m/z 428.13 involved cleavage of the phosphorus oxygen bond leading to ion at m/z
330.10. RS6 was the major degradation product of both oxidation and thermal stress,
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with increased retention compared to CoA. According to the result of HPLC and
LC-MS/MS, it is reasonable to speculate that to the thiol of CoA formed disulfide bond and the CoA was simultaneously added to an amino group. The mass
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chromatogram as well as the proposed structure and fragment pattern of RS5 were
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shown in Figure 8.
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3.2.7 RS7
RS7 exhibited [M+H]+ at m/z 769.1061 and [M+2H]2+ at m/z 385.0568 in LC-MS
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analysis. MS2 analysis showed product ions at m/z 508.17, which correspond to the
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cleavage of the phosphorus oxygen-bond and, produced a fragment at m/z 262.05. Another fragmentation pathway of the protonated molecules at m/z 508.17 concerned cleavage of the phosphodiester bond leading to the ion at m/z 427.93. RS7 was the
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major process-related substance with increased retention. In the light if the HPLC and LC-MS/MS data, it was reasonable to speculate that RS7 was formed by reaction of
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the product of the pantothenic acid phosphate ester and β-mercaptothion and triphosadenine during manufacturing. The mass chromatogram, proposed structure and fragment pattern of RS5 were shown in Figure 9.
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3.2.8 RS8
RS8 exhibited [M+H]+ at m/z 844.1209 and [M+H]2+ at m/z 422.5636 via MS analysis (Figure 10A). The MS2 analysis product ions was at m/z 508.17 which corresponded to cleavage of the phosphorus oxygen bond and produced fragments at m/z 336.99 and m/z 427.93 (Figure 10B). RS8 was not only a process-related substance, but also the major degradation product in acidic, alkaline, oxidation and
thermal stress with the decreased polarity. Combining the results of HPLC and LC-MS/MS, RS4 was generated by the reaction between CoA and β-mercaptothion. The fragment pattern of RS5 was shown in Figure 10C . 3.2.9 RS9 and RS10 The HPLC and LC-MS/MS results for RS9 and RS10 were consistent with coenzyme A disulfide reference and 3′-dephosphocoenzyme A reference, respectively.
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The mass chromatogram, proposed structure and fragment pattern of RS9 and RS10 were shown in Figure 11 and Figure 12, respectively.
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3.3 Synthesis and structural elucidation of RS4 3.3.1 Synthesis conditions
H2O2 (1%, 8 mL) was added to a stirred solution of coenzyme A (0.1 g) and L-Cys
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(0.1 g) dissolved in water (32 mL) and the mixture was stirred at 37 ºC for 3 h in a
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water bath. The reaction progress was monitored by HPLC (yield, 94%). The product
A
was purified by preparative HPLC (LC-20A) with a Shimadzu PRC-ODS (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium
M
acetate (pH 7.0) and methanol (94:6, v/v), with flow rate of 30 mL/min and UV
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detection was at 259 nm. The fractions of RS4 were lyophilized twice. HPLC detection verified the product purity (98.5%) (Figure S1). 3.3.2 Structural elucidation
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The 1H NMR spectral data for RS4 indicated that the number of hydrogen atoms agreed with the molecular formula of RS4 (Figure S2). The 13C NMR spectral data of
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RS4 showed that the line numbers were less than the carbon atoms which indicated the presence of carbon atoms with the same chemical shift. The 1H NMR and 13C NMR assignments for RS4 were summarized in table S1 and table S2. The NMR
A
spectra of RS4 were shown in Figure S3. 3.4 Synthesis and structural elucidation of RS5 3.4.1 Synthesis conditions Coenzyme A (0.1 g) was dissolved in hydrochloric acid (5 mL, 1 M) and the mixture was stirred at 37 ºC for 4 h in water bath. The reaction progress was monitored by HPLC (yield, 40%). The product was purified using preparative HPLC (LC-20A)
with an Ultimate XB-C18 (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium acetate (pH 7.0) and methanol (91:9, v/v), with a flow rate of 7 mL/min and UV detection at 259 nm. The fractions of RS5 were lyophilized twice. HPLC detection verified the product purity (96.6%) (Figure S1). 3.4.2 Structural elucidation The 1H NMR spectral data of RS5 indicated that the number of hydrogen atoms fit the
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RS5 molecular formula (Figure S2). The 13C NMR spectral data of RS5 showed that
line numbers were less than the carbon atoms which indicated the presence of carbon
were summarized in Table S3 and Table S4.
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atoms with same chemical shift. The 1H NMR and 13C NMR assignments for RS5
The NMR spectra of RS5 were shown
in Figure S4.
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3.5 Synthesis and structural elucidation of RS8
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3.5.1 Synthesis conditions
A
H2O2 (1%, 2 mL) and β-mercaptoethanol (50 μL) were added to a stirring solution of coenzyme A (0.1 g) dissolved in buffer (pH=8, 32 mL) and the mixture was stirred at
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37 ºC for 20 min in a water bath. The progress of the reaction was monitored by HPLC (yield, 95%). The product was purified by preparative HPLC (LC-20A) with a
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Shimadzu PRC-ODS (5 μm, 20×250 mm) column. The mobile phase consisted of 20 mM aqueous ammonium acetate (pH 7.0) and methanol (91:9, v/v), with flow rate of
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30 mL/min and UV detection was at 259 nm. The fractions of RS8 were lyophilized twice. HPLC detection verified the product purity (98.5%) (Figure S1).
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3.5.2 Structural elucidation The 1H NMR spectral data for RS8 indicated that the number of hydrogen atoms agreed with the RS8 molecular formula (Figure S2). The 13C NMR spectral data of
A
RS8 showed that line numbers were less than the carbon atoms which indicated thepresence of carbon atoms with the same chemical shift. The 1H NMR and 13C NMR assignments for RS4 were summarized in Table S5 and Table S6. The NMR spectra of RS8 were shown in Figure S5. 3.6 Optimization of HPLC method Initially, different types of HPLC columns, such as Agilent C8 (250 × 4.6 mm,5μm)
column, Agilent Eclipse Plus C18 (250 × 4.6 mm, 5μm) column, Agilent ZORBAX HILIC PLUS column (4.6 mm×100 mm, 3.5 μm) were tested to analysis CoA. The performance characteristics of the columns were mainly assessed for their ability to separate CoA and the related substances. The best resolution for the critical peaks was obtained on the Discovery Agilent Eclipse Plus C18 column which was thereafter used for further optimization of the method.
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Different gradient profile and pH values were explored all together to develop a
selective separation method. The retention of CoA was enhanced as pH decreased in
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the tested pH from 5.0-8.0 and the parameters and chromatogram were shown in
Figure S7 and Table S7. Shape symmetrical peaks with good resolution between CoA and its RSs were achieved at pH 7.0. The mechanism of CoA retention behavior vs.
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pH was proposed. The non-ionic proportion of CoA under acidic conditions was
N
greater than that under mild alkaline and neutral conditions due to the presence of the
A
adenine group in CoA. However, under acidic conditions, tailing peak appeared that was attributed to the ionic species of CoA that were simultaneously present in ionic
M
and non-ionic forms. Accordingly, the acid and alkaline conditions were not suitable
ED
for CoA analysis.
Different conditions of mobile phase were tested. The method has been optimized by comparing separation of related substance, shape symmetrical peaks of CoA and the
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number of related substances. Two gradient modes were developed with different initial proportions of mobile phase which named method-A (phase A: methanol/
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ammonium acetate aqueous =90/10, phase B: methanol) and method-B (phase A: methanol/ ammonium acetate aqueous=94/6, phase B: methanol). The gradient elution conditions were as follows: 0-30.0 min, linear from 0 to 25% B; 30.0-40.0 min
A
isocratic 0% B. The polar impurities cannot be well separated with method-A. However, a distorted peak of CoA was observed when decreased the initial proportion to 6% of organic phase (method-B). The separation of related substances was not satisfactory by a continuous gradient elution program. For the separation of RS5, RS6 and RS7 as well as the shape symmetrical peaks, the gradient profile was optimized. An isocratic hold process was applied in the method B to simultaneously improve the
separation and resolve the problem of distorted peak of CoA. Finally, the HPLC conditions were optimized by studying the effect of column types, buffer pH and gradient modes which was shown in section 2.2. 3.7 Validation of HPLC method 3.7.1 Quantitative method
concentration of 1 mg/mL for the analysis of related substances.
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Test solutions were accurately prepared by dissolving CoA in water to a final
Normally, three quantitative methods, area normalization method, main component
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self-compare method without a correction factor and main component self-compare method with a correction factor, were used to detect related substances. Correction factor is essential, given the difference responses of related substances and the
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principal component. In this experiment, three methods were applied to detect RS4,
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RS5, RS8 and RS10 and the result was shown in Table 2. The result showed that the
A
appropriate method was main component self-compare method. Therefore, the RS4, RS5, RS8 and RS10 were detected using the main component self-compare method
M
with correction factor and the other RSs were detected by main component
3.7.2 Specificity
ED
self-compare method without a correction factor.
The specificity of the method was checked using test solutions and the forced
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degradation solutions described in Section 2.5 using a DAD detector to determine the spectral peak purity of all of the chromatographic peaks. No interfering coeluting
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peaks were observed in the blank solutions, and the resolution between adjacent peaks was above 3.0 demonstrating adequate selectivity of the HPLC method (Figure 14). Assay studies were performed for stress samples against the CoA qualified reference
A
standards. The mass balance results were calculated for all of the stressed samples and found to be 99.3–101.3% at 259 nm, as shown in Table 3. 3.7.3 Limit of detection (LOD) and limit of quantitation (LOQ) LOD and LOQ were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively. The RSD of the areas for six replicate injections at the LOQ concentration were found to be below 10%, and the signal-to-noise ratios at LOD and LOQ concentration were
found to be below 3 and 10, respectively. The determined LOD values for RS4, RS5, RS8 and RS10 were 0.206, 0.084, 0.102 and 0.060 μg/mL and LOQ values were 0.514, 0.169, 0.203 and 0.124 μg/mL, respectively. The unknown impurities were represented by CoA and the LOD/LOQ was 0.060 and 0.18 μg/mL, respectively. 3.7.4 Linearity Linearity of the detector response was examined for the assay and related substances.
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For the CoA method, eight concentrations (0.45 μg/mL, 0.90 μg/mL, 1.8 μg/mL, 4.5 μg/mL, 9.0 μg/mL, 18 μg/mL, 45 μg/mL and 90 μg/mL) were prepared. For the
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related substances test RS4, RS5, RS8 and RS10 were examined by preparing
standard solution at eight different levels ranging from 0.5 μg/mL to 103 μg/mL, 0.42 μg/mL to 84 μg/mL, 0.51 μg/mL to 102 μg/mL, and 0.50 μg/mL to 100 μg/mL,
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respectively. The calibration equation, correlation coefficient (r) and correction factor
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of CoA and related substances were shown in Table 4 and the regression curves were
A
shown in Figure S6. 3.7.5 Accuracy
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Accuracy of the method was determined by spiking known impurities at 80%, 100%
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and 120% w/w of their specified limits in dosage form. The recovery of RS4, RS5, RS8 and RS10 ranged from 98.40%~103.48% (w/w) for the assay method and the relative standard deviation was less than 2%. The percentage recoveries were
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recorded in Table 4.
3.7.6 Precision and repeatability
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The reference substance CoA solution (10 μg/mL) was serially injected six times. The relative standard deviation of peak areas was 0.2%, showing good precision. Prepared six CoA solutions and their diluted solutions were injected to evaluate the
A
repeatability which was shown in Table 5. According to result, the RSD of content was less than 2%, indicating good repeatability for the method. 3.7.7 Stability of CoA solution Solution stability was tested by leaving spiked sample solutions in tightly capped volumetric flasks at 4 ℃ for 12 h. No significant change in peak areas was observed during the solution stability experiments. The results confirmed that the
solutions were stable for up to 12 h during the determination. The CoA solution (1 mg/mL) was tested after 24 h at 4 ℃. The RS3, RS4 and RS9 were unstable and the result of stability was shown in Table 6. Therefore, samples have to be prepared freshly before use. 3.7.8 Robustness To determine the robustness of the method, experimental conditions were deliberately
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changed: pH (7.0 ± 0.5), flow rate (1.0 ± 0.2 mL/min), detection wavelength (259 ± 5 nm), column temperature (25 ± 5 ℃) and different instruments (Agilent 1260 and
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Shimadzu 2010). Furthermore, mobile phase A was adjusted to 4% MET, 6% MET
(nominal) and 8% MET. The results obtained were assessed for system suitability to ensure that the separation requirements were maintained under the altered conditions
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and robustness was assessed by examination the content of related substances.
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Cumulative RSD was calculated for the RS5, RS8 and RS10 in the standard solution
A
and was found to be less than 10.0%. Results were summarized in Table 7. 3.8 Application of the method: analysis of commercial samples
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The proposed LC method was applied for the determination of related substances
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determination of CoA bulk drug (Sample A~I) available on the market. The results were summarized in Table 8. Among them, RS5, RS7, RS8, RS9 and RS10 were present at greater than 1.0%.
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4. Conclusion
A complex component of CoA was due to the microbial synthesis and the related
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substances of the highest contents in CoA were characterized. The main related substances in CoA were characterized by LC-MS/MS and product ions were predicted. Among them, RS4, RS5, and RS8 were further synthesized and elucidated by NMR
A
spectroscopy. In addition, a reliable HPLC method was developed and validated for quantitation analysis of related substances. The probable mechanism of formation of related substances was speculated. All the related substances came from the manufacturing process and some could be produced under the forced degradation conditions. Among them, RS2/6 were the major degradation products under oxidation stress, RS4/8/9 were the major degradation products under thermal degradation, RS5
was the major degradation product under acidic hydrolysis and RS8 was the major degradation product under alkaline hydrolysis. Related substance research is very important to the security, the quality promotion and assessment of drugs. The result reminds us to control the specific steps purposefully, as they are valuable for quality
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control in the manufacturing of CoA.
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Acknowledgment
This work was financially supported by Key Laboratory on Protein Chemistry and Structural Biology (No. 2016ZPT005), Supported by the Fundamental Research
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Appendix A. supplementary data
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The authors have declared no conflict of interest.
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Funds for the Central Universities (No. 2632017ZD07).
Supplementary data associated with this article described the NMR spectrograms and
References
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the MS spectrograms
A
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[1] F.P. Miller, A.F. Vandome, J. Mcbrewster, Coenzyme A, Alphascript Publishing 2010. [2] W.H.G. Devries, J.S. W. M. Evans, J.D. Gregory, G.D. Novelli, M. Soodak, F. Lipmann, Purification of coenzyme a from fermentation sources and its further partial identification, J. Am. Chem. Soc. 72 (1950) 4838-4838. [3] R.J. Perry, J.P.G. Camporez, R. Kursawe, P.M. Titchenell, D. Zhang, C.J. Perry, M.J. Jurczak, A. Abudukadier, M.S. Han, X.M. Zhang, H.B. Ruan, X. Yang, S. Caprio, S.M. Kaech, H.S. Sul, M.J. Birnbaum, R.J. Davis, G.W. Cline, K.F. Petersen, G.I. Shulman, Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes, Cell. 160 (2015) 745-758. [4] J.J. Kamphorst, M.K. Chung, J. Fan, J.D. Rabinowitz, Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate, Cancer. Metab. 2 (2014) 23-30. [5] L.G. Abo Alrob O, Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose
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oxidation. Biochem Soc Trans, Biochem. Soc. Trans. 42 (2014) 1043-1051. [6] A.M. Colombelli C, Tiranti V, Defective lipid metabolism in neurodegeneration with brain iron accumulation (NBIA) syndromes: not only a matter of iron, J. Inherited Metab. Dis. 38 (2015) 123-136. [7] T. Kupke, P. Hernándezacosta, F.A. Culiáñezmacià, 4'-phosphopantetheine and coenzyme A biosynthesis in plants, J. Biol. Chem. 278 (2003) 38229-38237. [8] J. Grünewald, Y. Jin, J. Vance, J. Read, X. Wang, Y. Wan, H. Zhou, W. Ou, H.E. Klock, E.C. Peters, Optimization of an Enzymatic Antibody-Drug Conjugation Approach Based on Coenzyme A Analogs, Bioconjugate Chem. 28 (2017) 1906-1915. [9] J.G. Nijssen, H.V.D. Bosch, Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes, Biochim. Biophys. Acta. 875 (1986) 458-464. [10] L. Jun, Clinical Application of Leucogen Combined with Coenzyme A in Treatment of Leukopenia, Chinese Journal of General Practice. 12 (2012) 1880-1911. [11] M. Takamiya, K. Saigusa, K. Dewa, DNA Microarray Analysis of the Mouse Adrenal Gland for the Detection of Hypothermia Biomarkers: Potential Usefulness for Forensic Investigation, Ther. Hypothermia. Tem. 3 (2013) 63-73. [12] M. G, Clinical trial of coenzyme A in hepatopathies, Minerva. Med. 67 (1976) 512-518. [13] M. Derot, M. Rathery, Coenzyme A in the treatment of chronic renal insuficiency, Therapie. 15 (1960) 603-611. [14] M. Derot, M. Rathery, The place of coenzyme A in the treatment of acute anuric renal insufficiency, Therapie. 15 (1960) 591-602. [15] M. Payet, M. Sankale, R. Camain, Y. Le Duc, Trial treatment of primary cancer of the liver by coenzyme A, Therapie. 15 (1960) 143-147. [16] M. Payet, M. Sankale, P. Pene, Y. Leduc, M. Moulanier, The treatment of lipoid nephrosis by coenzyme A, Therapie. 15 (1960) 217-230. [17] C. Amato, A. Majani, Paralytic ileus caused by pseudourological acute abdomen. Treatment with coenzyme A, Minerva chirurgica. 18 (1963) 683-686. [18] K. J, Impurity linked to adverse events, Chem. Eng. News. 86 (2008) 32-34. [19] W.B. Cong Luoluo, Guo Hongzhu, Shao Mingli, Data mining and association analysis of total related substance and adverse reaction rate of antibiotics, Zhongguo Xinyao Zazhi. 25 (2016) 964-967. [20] ICH guideline: Impurities in New Drug Substances Q3A (R2), 2006. [21] C. Fengyun, Determination of related substances in Coenzyme A with HPLC, Clin. Med. 25 (2005) 35-36. [22] L. Fuxiong, Determination of related substances in Coenzyme A for injection by gradient elution, Zhongguo Shenghua Yaowu Zazhi. 30 (2009) 124-126. [23] L. Ying, Study on the Quality Control for Coenzyme A, Pharmaceutical Sciences, Zhengzhou University, 2009. [24] ICH guidelines: Q1A (R2): Stability Testing of New Drug Substances and Products 2003.
Figure legends
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Figure 1. The synthesis processes of CoA. Figure 2. Chromatogram of Coenzyme A and ten related substances. Figure 3. The mass chromatogram and plausible scheme for fragmentations of CoA. Figure 4. The mass chromatogram and plausible scheme for fragmentations of RS2. Figure 5. The mass chromatogram and plausible scheme for fragmentations of RS3. Figure 6. The mass chromatogram and plausible scheme for fragmentations of RS4. Figure 7. The mass chromatogram and plausible scheme for fragmentations of RS5. Figure 8. The mass chromatogram and plausible scheme for fragmentations of RS6. Figure 9. The mass chromatogram and plausible scheme for fragmentations of RS7. Figure 10. The mass chromatogram and plausible scheme for fragmentations of RS8. Figure 11. The mass chromatogram and plausible scheme for fragmentations of RS9. Figure 12. The mass chromatogram and plausible scheme for fragmentations of RS10. Figure 13. Probable mechanism of formation of RS2~RS10. Figure 14. Typical HPLC chromatograms of CoA and its degradation products formed under (A) 2.0 M HCl, 37 ºC, 1 h, (B) 2 M NaOH, 37 ºC, 1 h, (C) 1% H2O2, 37 ºC, 1 h, (D) H2O, 60 ºC, 2 h, (E) natural light, 6 h.
A ED
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SC R
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N
A
M
A ED
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SC R
U
N
A
M
A ED
PT
CC E
IP T
SC R
U
N
A
M
A ED
PT
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U
N
A
M
A ED
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N
A
M
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A
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Table 1 TOF mass spectra results of CoA and its related substances. Theoretical
Proposed
Observed ion related
tR (min)
Error ion mass
Molecular
Product ions
(m/z)
Formula [M+H]
768.1225
C21H37N7O16P3S
---
---
mass (m/z) substances CoA
8.81
768.1246
(ppm)
RS1
2.38
353.0834,
2.73
428.09, 260.68
SC R
337.0029,
+
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Name of
---
3.29
816.1095
816.1072
C21H37N7O19P3S
2.82
428.36, 389.26, 309.18
RS3
4.60
812.1130
812.1123
C22H37N7O18P3S
0.86
428.12, 305.24
RS4
6.47
887.1276
887.1266
C24H42N8O18P3S2
1.13
379.45, 330.01
RS5
10.30
768.1224
768.1225
C21H37N7O16P3S
-0.13
428.04, 260.91
RS6
11.02
843.1360
843.1368
C23H42N8O16P3S2
-0.95
428.13, 336.42, 330.10
RS7
11.91
769.1061
769.1065
C21H36N6O17P3S
-0.52
508.17, 427.93, 262.05
RS8
17.85
844.1209
844.1208
C23H41N7O17P3S2
0.12
508.17, 427.93. 336.99
767.1146
[C42H72N14O32P6S2]2+
0.00
617.92, 519.99, 428.58
A
RS10
19.52
20.34
M
ED
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CC E
RS9
767.1146
688.1558
A
RS2
N
U
403.0299
---
513.64, 428.22, 688.1562
C21H36N7O13P2S
-0.58 348.48, 261.17
Table 2 Compare the results of RS contents by different quantitative methods
Content o Content
Content o Content o um Co
Total cont
f CoA
f
ent of RS
f
ntent o
RS5（%） RS8（%） RS10（%） f RS
SC R
（%）
of
4.3
U
Quantitative method
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Maxim
3.51
4.5
0.65
3.06
（%）
（%）
7
Main-component self-co ction factor
0.14 4
Main-component self-co mpare method with correcti
4 0.17
5
PT CC E A
5 4.55
2
3.34
7
0.53
1
5.65
ED
on factor
1
0.50
A
mpare method without corre
4.33
M
method
0.13
N
Area normalization
0
31
9
5
15.30 3
54
16.16 9
5.6 52
16.96 3
Table 3 The parameters of forced degradation studies.
Resolution between CoA Cont
Samples
ent（%）
CoA peak and nearest p
Mass bala nce （%）
Peak puri ty of CoA
ysis Oxidation degr adation Thermal degrad ation Light degradati
3.2
100.5
48.195
3.7
72.181
4.0
85.454
3.3
85.312
3.9
A
CC E
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ED
M
on
68.580
SC R
Alkaline hydrol
——
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sis
4.0
N
Acidic hydroly
85.018
A
Sample
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eak 0.979 0.948
100.8
0.990
101.3
0.986
99.3
0.967
99.4
0.984
Table 4 Linearity and recovery data.
Analyte
Calibration
Correlation
Correction
equation
coefficient (r)
factor
Recovery (%)
recovery
1
---
RS 4
y=15426x-8852.1
0.9999
1.22
RS 5
1
1.24
1
RS 10
y=21551x-6824.8
1
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*the correction factor is slope CoA/slope RS
A
99.637
0.812
99.813
0.903
102.791
0.565
1.22
A
y=15324x-5660.0
M
RS 8
1.164
N
U
y=15120x-4885.5
101.828
SC R
y=18766x-4408.8
---
IP T
--CoA
RSD of
0.87
Table 5 The repeatability of related substances.
RSD (%, n=6)
RS2
0.598
1.1
RS3
0.621
0.7
RS4
0.076
1.5
RS5
5.152
0.2
RS6
0.260
0.1
RS7
1.977
0
RS8
0.616
RS9
0.833
A
CC E
PT
Oher RS
2.293
SC R
U
N 0.1
A
M 2.947
ED
RS10
IP T
Content（%） Average
1.2 0.1 0.6
Table 6 Stability data. Content of CoA
Content of RS3
Content of RS4
Content of RS9
（%）
（%）
（%）
（%）
Room
Room
Room
Room
（h）
re
4
84.825
0.816
84.91
0.86 0.858
0.10
0.122
5
84.959
85.06
0.88
ED
8
M
0.88
84.96
3 0.66
0.685 9
1
0.11
0.66
0.139
0.731 1
4
0.90
0.11
0.66
0.168
0.774
3
1
3
7
85.00
0.92
0.11
0.67
0.918
0.184
0.818
9
5
7
84.93
0.95
0.12
84.564
A
24
0.65
5
0.892
84.774
CC E
16
84.82
PT
12
4℃
0.653
7
A
8
temperatu
0.10
0.107 6
4℃
re
0.81
5
84.916
temperatu re
84.82 0
4℃
SC R
re
temperatu
U
4℃
N
temperatu
IP T
Time
0.924 4
0.196 6
8
0.896
0.69
11.8
2
8
RSD 0.2 （%）
0.1
4.6
5.5
23.3
6.7
Table 7 The data of robustness RSD of concentration (%) RS10
A
2.43
2.67
3.89
B
1.86
0.48
2.10
C
2.68
3.82
1.81
D
1.09
7.35
0.35
E
1.37
0.86
1.73
F
1.01
1.24
IP T
RS8
SC R
RS5
U
Conditions
N
0.83
A
*A: pH (7.0 ± 0.5); B: initial proportion of phase A (4% MET, 6% MET and 8% MET); C:
M
instruments (Agilent 1260 and Shimadzu 2010); D: column temperature (25 ± 5 ℃); E:
A
CC E
PT
ED
flow rate (1.0 ± 0.2 mL/min); F: detection wavelength (259 ± 5 nm).
Table 8 Results of impurity determination for CoA drug substance samples (A-I).
Sample
IP T
Content (%) Other
RS3
RS4
RS5
RS6
RS7
RS8
RS9
SC R
RS2
RS10
RSs
0.202
0.675
0.097
3.751
c
1.572
0.737
1.088
2.911
1.063
Sample B
0.084
0.579
0.11
5.293
0.197
2.381
0.71
0.605
2.574
1.308
Sample C
0.074
0.612
0.126
3.875
0.165
Sample D
0.164
0.665
0.16
4.126
Sample E
0.169
0.857
0.076
Sample F
0.546
0.889
0.175
0.478
0.857
0.58
CC E
Sample H
0.759
0.556
2.338
0.772
0.176
1.447
0.943
0.582
4.041
1.028
3.922
0.151
2.43
1.979
0.567
6.626
1.863
5.652
0.258
2.058
0.65
0.863
3.065
2.806
M
A
N
1.492
ED
G
0.168
5.304
0.246
1.938
0.605
0.852
2.9
2.847
0.87
0.179
5.379
0.253
1.969
0.627
0.819
2.934
2.698
0.867
0.195
5.205
0.16
1.063
2.47
1.34
1.456
1.56
PT
Sample
U
Sample A
Sam
0.375
A
ple I