Development and validation of a multiplex UHPLC‐MS/MS assay with stable isotopic internal standards for the monitoring of the plasma concentrations of the antiretroviral drugs bictegravir,cabotegravir, doravirine,and rilpivirine in people living with HIV

The widespread use of highly active antiretroviral treatments has dramatically changed the prognosis of people living with HIV (PLWH). However, such treatments have to be taken lifelong raising issues regarding the maintenance of both therapeutic effectiveness and long‐term tolerability. Recently approved or investigational antiretroviral drugs present considerable advantages, allowing once daily oral dosage along with activity against resistant variants (eg, bictegravir and doravirine) and also parenteral intramuscular administration that facilitates treatment adherence (eg, long‐acting injectable formulations such as cabotegravir and rilpivirine). Still, there remains a risk of insufficient or exaggerated circulating exposure due to absorption issues, abnormal elimination, drug‐drug interactions, and others. In this context, a multiplex ultra‐high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS) bioassay has been developed for the monitoring of plasma levels of bictegravir, cabotegravir, doravirine, and rilpivirine in PLWH. A simple and convenient protein precipitation was performed followed by direct injection of the supernatant into the UHPLC‐MS/MS system. The four analytes were eluted in less than 3 minutes using a reversed‐phase chromatography method coupled with triple quadrupole mass spectrometry detection. This bioassay was fully validated following international guidelines and achieved good performances in terms of trueness (94.7%‐107.5%), repeatability (2.6%‐11%), and intermediate precision (3.0%‐11.2%) over the clinically relevant concentration ranges (from 30 to 9000 ng/mL for bictegravir, cabotegravir, and doravirine and from 10 to 1800 ng/mL for rilpivirine). This sensitive, accurate, and rapid UHPLC‐MS/MS assay is currently applied in our laboratory for routine therapeutic drug monitoring of the oral drugs bictegravir and doravirine and is also intended to be applied for the monitoring of cabotegravir/rilpivirine levels in plasma from PLWH receiving once monthly or every 2‐month intramuscular injection of these long‐acting antiretroviral drugs.     

Analysis of impurities in pharmaceuticals by LC‐MS with cold electron ionization

Pharmaceuticals require careful and precise determination of their impurities that might harm the user upon consumption. Although today, the most common technique for impurities identification is liquid chromatography‐mass spectrometry (LC‐MS/MS), it has several downsides due to the nature of the ionization method. Also, the analyses in many cases are targeted thus despite being present, some of the compounds will not be revealed. In this paper, we propose and show a new method for untargeted analysis and identification of impurities in active pharmaceutical ingredients (APIs). The instrument used for these analyses is a novel electron ionization (EI) LC‐MS with supersonic molecular beams (SMB). The EI‐LC‐MS‐SMB was implemented for analyses of several drug samples spiked with an impurity. The instrument provides EI mass spectra with enhanced molecular ions, named Cold EI, which increases the identification probabilities when the compound is identified with the aid of an EI library like National Institute of Standards and Technology (NIST). We analyzed ibuprofen and its impurities, and both the API and the expected impurity were identified with names and structures by the NIST library. Moreover, other unexpected impurities were found and identified proving the ability of the EI‐LC‐MS‐SMB system for truly untargeted analysis. The results show a broad dynamic range of four orders of magnitude at the same run with a signal‐to‐noise ratio of over 10 000 for the API and almost uniform response.     

新型除草剂及其杂质的LC-MS和GC-MS分析

High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) have been utilized to analyze the synthesized 2-(2-arylaminomethylphenoxy)pyrimidine derivatives, which are a new kind of environmentally benign herbicides and have passed the temporary pesticide registration. The identification of main product and impurities has been achieved according to the UV and mass spectra. Moreover, one impurity, introduced by the raw material in the last step of the synthetic route, was identified by GC-MS analysis. It can be concluded that the combination of chromatography and mass spectrometry, including LC-MS and GC-MS, provided a vital tool of the pesticide science.     

Semi‐preparative LC‐SPE‐cryoflow NMR for impurity identifications: use of mother liquor as a better source of impurities

Unambiguous structural elucidation of active pharmaceutical ingredients (API) impurities is a particularly challenging necessity of pharmaceutical development, particularly if the impurities are low level (0.1% level). In many cases, this requires acquiring high‐quality NMR data on a pure sample of each impurity. High‐quality, high signal‐to‐noise (S/N) one‐ and two‐dimensional NMR data can be obtained using liquid chromatography‐solid phase extraction‐cryoflow NMR (LC‐SPE‐cryoflow NMR) with a combination of semi‐preparative column for separation and mother liquor as a source of concentrated impurities. These NMR data, in conjunction with mass spectrometry data, allowed for quick and unambiguous structural elucidations of four impurities found at low level in the crystallized API but found at appreciable levels in the mother liquor that was used as the source for these impurities. These data show that semi‐preparative columns can be used at lower than ideal flow rates to facilitate trapping of HPLC components for LC‐SPE‐cryoflow NMR analysis without compromising chromatographic resolution. Also, despite the complex chromatography encountered with the use of mother liquor as a source of impurities, acceptably pure analytes were obtained for acquiring NMR data for unambiguous structure elucidations. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of major degradation products of an adenosine A2A receptor antagonist under stressed conditions by LC‐MS and FT tandem MS analysis

Parkinson's disease (PD) is a very serious neurological disorder, and current methods of treatment fail to achieve long‐term control. SCH 420814 is a potent, selective and orally active adenosine A_2A receptor antagonist discovered by Schering‐Plough. Stability testing provides evidence of the quality of a bulk drug when exposed to the influence of environmental factors. Understanding the drug degradation profiles is critical to the safety and potency assessment of the drug candidate for clinical trials. As a result, identification of degradation products has taken an important role in drug development process. In this study, a rapid and sensitive method was developed for the structural determination of the degradation products of SCH 420814 formed under different forced conditions. The study utilizes a combination of liquid chromatography–tandem‐mass spectrometry (LC‐MS/MS) and Fourier Transform (FT) MS techniques to obtain complementary information for structure elucidation of the unknowns. This combination approach has significant impact on degradation product identification. A total of ten degradation products of SCH 420814 were characterized using the developed method. Copyright © 2009 John Wiley & Sons, Ltd.     

Challenges in the identification process of phenidate analogues in LC‐ESI‐MS/MS analysis: Information enhancement by derivatization with isobutyl chloroformate

A new analytical technique for the structural elucidation of four representative phenidate analogues possessing a secondary amine residue, which leads to a major/single amine‐representative fragment/product ion at m/z 84 both in their GC‐EI‐MS and LC‐ESI‐MS/MS spectra, making their identification ambiguous, was developed. The method is based on “in vial” chemical derivatization with isobutyl chloroformate in both aqueous and organic solutions, followed by liquid chromatography‐electrospray ionization mass spectrometry (LC‐ESI‐MS/MS). The resulting carbamate derivatives promote rich fragmentation patterns with full coverage of all substructures of the molecule, enabling detailed structural elucidation and unambiguous identification of the original compounds at low ng/mL levels.     

Applications of LC/MS in structure identifications of small molecules and proteins in drug discovery

With advancements in ionization methods and instrumentation, liquid chromatography/mass spectrometry (LC/MS) has become a powerful technology for the characterization of small molecules and proteins. This article will illustrate the role of LC/MS analysis in drug discovery process. Examples will be given on high-throughput analysis, structural analysis of trace level impurities in drug substances, identification of metabolites, and characterization of therapeutic protein products for process improvement. Some unique MS techniques will also be discussed to demonstrate their effectiveness in facilitating structural identifications.     

Identification and characterization of stressed degradation products of metoprolol using LC/Q-TOF-ESI-MS/MS and MS(n) experiments

A rapid, specific and reliable isocratic high-performance liquid chromatography combined with quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) method has been developed and validated for the identification and characterization of stressed degradation products of metoprolol. Metoprolol, an anti-hypertensive drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress, as per ICH-specified conditions. The drug showed extensive degradation under oxidative and hydrolysis (acid and base) stress conditions. However, it was stable to thermal, neutral and photolysis stress conditions. A total of 14 degradation products were observed and the chromatographic separation of the drug and its degradation products was achieved on a C(18) column (4.6 × 250 mm, 5 μm). To characterize degradation products, initially the mass spectral fragmentation pathway of the drug was established with the help of MS/MS, MS(n) and accurate mass measurements. Similarly, fragmentation pattern and accurate masses of the degradation products were established by subjecting them to LC-MS/QTOF analysis. Structure elucidation of degradation products was achieved by comparing their fragmentation pattern with that of the drug. The degradation products DP(2) (m/z 153) and DP(14) (m/z 236) were matched with impurity B, listed in European Pharmacopoeia and British Pharmacopoeia, and impurity I, respectively. The LC-MS method was validated with respect to specificity, linearity, accuracy and precision.     

MSn,LC‐MS‐TOF and LC‐PDA studies for identification of new degradation impurities of bupropion

Three new degradation impurities of bupropion were characterized through high performance liquid chromatography coupled to photodiode array detection and to time‐of‐flight mass spectrometry. Bupropion was subjected to the ICH prescribed stress conditions. It degraded to seven impurities (I–VII) in alkaline hydrolytic conditions which were optimally resolved on an XTerra C₁₈ column (250 × 4.6 mm, 5 µm) with a ternary mobile phase comprising ammonium formate (20 mm , pH 4.0), methanol and acetonitrile (75:10:15, v/v). The degradation impurities (III–V and VII) were characterized on the basis of mass fragmentation pattern of drug, accurate mass spectral and photodiode array data of the drug and degradation impurities. Compound V was found to be a known degradation impurity [1‐hydroxy‐1‐(3‐chlorophenyl)propan‐2‐one], whereas III, IV and VII were characterized as 2‐hydroxy‐2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorpholine, (2,4,4‐trimethyl‐1,3‐oxazolidin‐2‐yl)(3‐chlorophenyl)‐methanone and 2‐(3′‐chlorophenyl)‐3,5,5‐trimethylmorphol‐2‐ene, respectively. Compound III was a known metabolite of the drug. This additional information on the degradation impurities can help in the development of a new stability‐indicating assay method to monitor the stability of the drug product during its shelf‐life as well as in development of a drug product with increased shelf‐life. Copyright © 2013 John Wiley & Sons, Ltd.     

Applications of hyphenated LC-MS techniques in pharmaceutical analysis

In pharmaceutical analysis, ie the analytical development and quality control of drug substances and dosage forms, mass spectrometry (MS) combined with chromatographic separation is perhaps the most powerful technique for the monitoring, characterization and identification of impurities. The addition of further dimensions to chromatographic separations by hyphenated techniques offers unique possibilities of efficiently supporting pharmaceutical development and ensuring the quality and safety of pharmaceuticals. However, the ionization process in MS involves some characteristics which have to be recognized and taken into account for an appropriate application as well as the evaluation of the results. Chromatographic method development and validation can be supported very effectively by MS detection, eg in the investigation of coelution and peak purity. Chiral amino acid analysis is largely facilitated by the mass-specific detection of the derivatized amino acid enantiomers, which ignores all other interfering substance peaks. Examples are presented for the use of LC-MS-MS fragmentation and high-resolution MS in the structural elucidation of degradation products and impurities. LC-MS is systematically applied to monitor impurity profiles during pharmaceutical development and scaling up and supports the safety evaluation of batches used in clinical studies.     

Assessment and validation of the MS/MS fragmentation patterns of the macrolide immunosuppressant everolimus

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.     

Application of two‐dimensional selective‐TOCSY HMBC for structure elucidation of impurities in mixture without separation

In the pharmaceutical industry, regulatory expectations driven by patient safety considerations make structure elucidation of impurities at levels greater than 0.1% in the active pharmaceutical ingredient (API) of primary interest. Impurities can be generated from isomers in starting materials, or produced from different process steps toward the final API. Proton peaks belonging to different impurities could be potentially identified in the one‐dimensional ¹H NMR spectrum, when evaluated in combination with two‐dimensional (2‐D) COSY and HSQC data. However, in 2‐D HMBC data, correlation responses from different impurities may overlap with those from the major component, causing uncertainty of long‐range proton to carbon correlations and quaternary carbon assignments. This observation prompts us to design the 2‐D selective‐TOCSY HMBC experiment to distinguish responses from different impurities in mixtures to obtain 2‐D NMR data for each impurity, thus eliminating the use of a chromatographic isolation step to obtain material for NMR analysis. This methodology is demonstrated for structure elucidation of impurities ranging from 8.2% in the raw material to 0.4% in the API in this study, and would be particularly useful for industrial samples in which the solubility and availability of material are not an issue. Copyright © 2012 John Wiley & Sons, Ltd.     

Fragmentation patterns study of iridoid glycosides in Fructus Gardeniae by HPLC‐Q/TOF‐MS/MS

Iridoid glycosides (IGs), the major constituents in Fructus Gardeniae, have demonstrated various pharmacological activities, but there is no systematic chemical profile of IGs in Fructus Gardeniae in the published literature until now. Therefore, it is imperative that a rapid and sensitive high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐Q/TOF‐MS/MS) method is established for comprehensive characterization of IGs in Fructus Gardeniae. Firstly, the fragmentation patterns of six known IGs were investigated and proposed and further concluded the diagnostic fragment ions and characteristic fragmentation pathways. Then, based on the summarized fragmentation patterns and the known compounds in the literatures, the other IGs in Fructus Gardeniae were identified successively. As a result, a total of 20 IGs were identified, of which three pairs of epimers were structurally characterized and differentiated. More importantly, one compound, the isoshanzhiside methyl ester, was tentatively identified as a new compound. The results of this study demonstrate the superiority of HPLC‐MS with a high‐resolution mass spectrometer for the rapid and sensitive structural elucidation of the multiple groups of constituents in Fructus Gardeniae. Copyright © 2014 John Wiley & Sons, Ltd.     

液相色谱-串联质谱法测定吉非替尼中4种基因毒性杂质

建立了一种同时测定吉非替尼中4种基因毒性杂质3-氯-4-氟苯胺、3,4-二氟苯胺、3-氟-4-氯苯胺和3,4-二氯苯胺的高效液相色谱-串联质谱（HPLC-MS/MS）方法。用Inertsil ODS-3柱（100 mm×3.0 mm,3μm）为色谱柱,以0.1%（体积分数,下同）甲酸水溶液-0.1%甲酸乙腈溶液为流动相,在电喷雾正离子模式下进行测定。该方法在特异性、线性、精密度、准确性、稳定性和耐用性方面得到了验证。4种基因毒性杂质在0.6~96.0μg/L范围内与峰面积呈良好线性关系。检测限和定量限分别为0.2~2.0μg/L和0.6~6.0μg/L。所有杂质的回收率为91.0%~98.5%。检测后,在批号16052301和R16052501-1样品中仅检测到3-氯-4-氟苯胺,但低于杂质限度（6 mg/L）。该方法简便可靠,可用于吉非替尼中4种基因毒性杂质的测定,并为质量控制提供参考。     

Identification,characterization, and high‐performance liquid chromatography quantification of process‐related impurities in vonoprazan fumarate

High‐performance liquid chromatography analysis of vonoprazan fumarate, a novel proton pump inhibitor drug revealed six impurities. These were identified by liquid chromatography with mass spectrometry. Further, the structures of the impurities were confirmed by synthesis followed by characterization by mass spectrometry, NMR spectroscopy, and infrared spectroscopy. On the basis of these data and knowledge of the synthetic scheme of vonoprazan fumarate, the previously unknown impurity was identified as 1‐[5‐(2‐fluorophenyl)‐1‐(pyridin‐3‐ylsulfonyl)‐1H‐pyrrol‐3‐yl]‐N‐methyldimethylamine, which is a new compound. The possible mechanisms by which these impurities were formed were also discussed. A high‐performance liquid chromatography method was optimized in order to separate, selectively detect, and quantify all process‐related impurities of vonoprazan fumarate. The presented method has been validated in terms of linearity, limits of detection, and quantification, and response factors and, therefore, is highly suitable for routine analysis of vonoprazan fumarate related substances as well as stability studies.     

Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites in urine by LC‐MS after derivatization and its application for differentiation between positional isomers of prilocaine

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide‐type local anesthetic drugs and their main metabolites by GC‐EI‐MS and LC‐ESI‐MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine‐containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential “in‐vial” instantaneous derivatization processes at ambient temperature followed by LC‐ESI‐MS/MS analysis was developed. 2,2,2‐Trichloro‐1,1‐dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI‐MS and ESI‐MS/MS spectra, enabling their structural elucidation in a single LC‐ESI‐MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.     

Assay determination by mass spectrometry for oligonucleotide therapeutics

Phosphorothioate oligonucleotide drugs typically contain product‐related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co‐eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry‐based purity assessment of the main chromatography peak can be used to quantify co‐eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co‐eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co‐sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry‐based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R² = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications.     

Rapid detection and identification of impurities in ten 2-naphthalenamines using an atmospheric pressure solids analysis probe in conjunction with ion mobility mass spectrometry

The atmospheric pressure solids analysis probe (ASAP), in conjunction with ion mobility time-of-flight mass spectrometry (IM-ToF-MS), has been applied to the impurity profiling study of ten 2-naphthalenamines. The impurity profiles achieved by ASAP-IM-MS were compared with those obtained by liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). All the impurities at the level of 0.1 area % and above, except for one, detected by LC-ESI-MS, were also found by ASAP-IMS analyses. In addition, one non-polar compound was detected by ASAP-IM-MS alone. The IM-MS plot of ion drift time versus m/z values offered sufficient separation between the impurities with different m/z. Therefore, instead of LC as a separation tool, IM-MS is able to provide fingerprint profiling for the ten samples analysed. The time of each analysis has been reduced from 25 min by LC-MS to less than 3 min by ASAP-IM-MS. When collision energy was applied for the selected precursor ion in the transfer T-wave, a clean MS/MS spectrum was obtained for structural elucidation of unknown impurities. The hyphenation of ASAP and IM-MS techniques represents a highly efficient approach for rapid detection and identification of impurities generated in complex reactions involved in pharmaceutical development.     

Qualitative and quantitative analysis of major constituents from Dazhu Hongjingtian capsule by UPLC/Q‐TOF‐MS/MS combined with UPLC/QQQ‐MS/MS

In this work, a sensitive and efficient method was established and validated for qualitative and quantitative analysis of major bioactive constituents in Dazhu Hongjingtian capsule by liquid chromatography tandem mass spectrometry. A total of 32 compounds were tentatively identified using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Furthermore, 12 constituents, namely gallic acid, 3,4‐dihydroxybenzoic acid, salidroside, p‐ coumaric acid‐4‐O ‐β ‐d ‐glucopyranoside, bergeninum, 4‐hydroxybenzoic acid, 4‐hydroxyphenylacetic acid, syringate, 6′′‐O ‐galloylsalidroside, rhodiosin, rhodionin and kaempferol‐7‐O ‐α ‐l ‐rhamnoside, were simultaneously quantified by the developed ultra‐performance liquid chromatography coupled with a triple quadrupole mass spectrometry method in 9 min. All of them were analyzed on an Agilent ZorBax SB‐C₁₈ column (3.0 × 100 mm, 1.8 μm) with linear gradient elution of methanol–0.1% formic acid water. The proposed method was applied to analyze three batches of samples with acceptable linearity (R , 0.9979–0.9997), precision (RSD, 1.3–4.7%), repeatability (RSD, 1.7–4.9%), stability (RSD, 2.2–4.9%) and recovery (RSD, 0.6–4.4%) of the 12 compounds. As a result, the analytical method possessing high throughput and sensitivity is suitable for the quality control of Dazhu Hongjingtian capsule.