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Pharmaceutical regulatory agencies are increasingly concerned with trace-level genotoxic impurities in drug substances, requiring manufacturers to deliver innovative approaches for their analysis and control. The need to control most genotoxic impurities in the low ppm level relative to the active pharmaceutical ingredient (API), combined with the often reactive and labile nature of genotoxic impurities, poses significant analytical challenges. Therefore, sophisticated analytical methodologies are often developed to test and control genotoxic impurities in drug substances. From a quality-by-design perspective, product quality (genotoxic impurity levels in this case) should be built into the manufacturing process. This necessitates a practical analysis and control strategy derived on the premise of in-depth process understanding. General guidance on how to develop strategies for the analysis and control of genotoxic impurities is currently lacking in the pharmaceutical industry. In this work, we demonstrate practical examples for the analytical control of five genotoxic impurities in the manufacturing process of pazopanib hydrochloride, an anticancer drug currently in Phase III clinical development, which may serve as a model for the other products in development. Through detailed process understanding, we implemented an analysis and control strategy that enables the control of the five genotoxic impurities upstream in the manufacturing process at the starting materials or intermediates rather than at the final API. This allows the control limits to be set at percent levels rather than ppm levels, thereby simplifying the analytical testing and the analytical toolkits to be used in quality control laboratories.

Derivatization LC/MS methodology has been developed for the determination of a group of commonly encountered alkyl esters of sulfonates or sulfates in drug substances at low ppm levels. This general method uses trimethylamine as the derivatizing reagent for ethyl/propyl/isopropyl esters and triethylamine for methyl esters. The resulting quaternary ammonium derivatization products are highly polar (ionic) and can be retained by a hydrophilic interaction liquid chromatography (HILIC) column and readily separated from the main interfering active pharmaceutical ingredient (API) peak that is usually present at very high concentration. The method gives excellent sensitivity for all the alkyl esters at typical target analyte level of 1-2 ppm when the API samples were prepared at 5mg/mL. The recoveries at 1-2 ppm were generally above 85% for all the alkyl esters in the various APIs tested. The injection precisions of the lowest concentration standards were excellent with R.S.D.=0.4-4%. A linear range for concentrations from 0.2 to 20 ppm has been established with R(2)>or=0.99. This general method has been tested in a number of API matrices and used successfully for determination of alkyl sulfonates or dialkyl sulfates in support of API batch releases at GlaxoSmithKline.

Structural elucidation of pharmaceutical impurities in drug substances and drug products is an important task in pharmaceutical analysis in various phases of drug development. Liquid chromatography-mass spectrometry (LC-MS) technologies play a key role in this task owing to their general attributes of superior selectivity, sensitivity and speed. Full scan and product ion scan analysis, providing molecular weight information and fragmentation data, respectively, offer rich structural information and allow proposal of candidate structures rather quickly. However, these proposed structures often lack certainty especially when dealing with structural isomers. On-line hydrogen/deuterium (H/D) exchange by LC-MS using D2O as the mobile phase component is a powerful tool for identifying active hydrogen atoms, thus constituting a simple strategy for distinguishing between isomeric structures which are sometimes difficult by product ion spectral data or accurate mass data. This review describes the typical experimental setup we use routinely in the laboratories for performing H/D exchange LC-MS experiments in conjunction with representative applications of the strategy in structural elucidation of pharmaceutical impurities.

Strategies using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conjunction with techniques such as chemical derivatization and on-line hydrogen/deuterium (H/D) exchange for structural elucidation of drug metabolites in crude samples are reviewed. Useful mass spectrometric scan techniques discussed include product ion scan, constant neutral-loss scan, precursor ion scan, multistage MS(n), and accurate mass measurements. In biological systems, xenobiotics are transformed into metabolites, which usually involves introduction of one or more polar functional groups or removal or blockage of such structural moieties. Therefore, chemical derivatization strategies for determination of functional groups and on-line H/D exchange approaches for probing number of exchangeable hydrogens are powerful tools for structural elucidation of drug metabolites in drug metabolism studies. More importantly, these experiments can be carried out on crude samples in microscale, providing sufficient material for LC-MS/MS analysis. Therefore, labor intensive and technically challenging purification of low levels of drug metabolites from complex biological matrices can be avoided. It is the authors' conclusion that strategies such as chemical derivatization and on-line H/D exchange should be used more routinely in drug metabolism studies in order to facilitate metabolite identification.

The in vitro metabolism of MK-0767 [(+/-)-5-[(2,4-dioxothiazolidin-5-yl) methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a novel 2,4-thiazolidinedione (TZD)-containing peroxisome proliferator-activated receptor alpha/gamma agonist, was studied in rat, dog, monkey, and human liver microsomes and hepatocytes, as well as in recombinant human CYP3A4-containing microsomes. Twenty-two metabolites (some at trace levels) were detected by liquid chromatography-tandem mass spectrometry analysis. All appeared to be phase I metabolites except for a glucuronide conjugate of a hydroxylated metabolite that was detected at trace levels. A constant neutral loss scan experiment performed on a triple quadrupole mass spectrometer proved to be very useful for resolving the metabolites from endogenous compounds. It was observed that the initial site of metabolism of MK-0767 was at the TZD ring leading to two major metabolites, namely the 5-hydroxy-TZD metabolite (M24) and the mercapto metabolite (M22). The latter was formed via the cleavage of the TZD ring with the elimination of the carbonyl adjacent to the sulfur atom. The structure of M24 was established by accurate mass measurements and NMR analysis. This hydroxy-TZD metabolite might represent an important precursor for a group of metabolites formed by TZD ring opening and subsequent loss of the sulfur moiety. The mercapto metabolite, on the other hand, is probably the key precursor for the TZD ring-opened metabolites with retention of the sulfur, even though the detailed mechanism of the ring scission remains to be characterized. From these studies, it was concluded that the TZD ring was the major site of metabolism of MK-0767. All the metabolites produced in vitro from human preparations were detected in the corresponding preparations from the nonclinical species.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%. Copyright © 2001 John Wiley & Sons, Ltd.

Elesclomol (N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide)), which is a novel anticancer agent, can form chelating complexes with Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) in the gas phase during electrospray ionization (ESI) mass spectrometry. In the solution phase with acidic medium during chromatographic separation, however, only Cu(II) and Ni(II) to a lesser degree favor the formation of chelating complexes with elesclomol. The Cu(II)-chelating complex [Cu(II)+elesclomol-H]+· exhibits more complicated MS/MS fragmentation pathways than the Ni(II)-chelating complex [Ni(II)+elesclomol-H]+. One significant difference is the ready occurrence of the electron transfer upon collision-induced dissociation (CID) of [Cu(II)+elesclomol-H]+·. This leads to the reduction of Cu(II) to Cu(I). However, such phenomenon was not observed upon CID of [Ni(II)+elesclomol-H]+. On the basis of the density functional theory (DFT) calculations at the B3LYP/6-31+G(d)/LANL2DZ level, the Cu(II)- and Ni(II)-chelating complexes of elesclomol exist in the keto-form with tetra-coordinated trapezoid geometry in the gas phase but at different levels of distortion. As compared to the Ni(II)-elesclomol complex, the Cu(II)-elesclomol complex is more stable (by -55.25 kcal/mol). This relative stability of the chelating complexes of elesclomol is consistent with the Irving-Williams series of bindings to ligands.

Trace analysis of unstable and reactive pharmaceutical genotoxic impurities (GTIs) is a challenging task in pharmaceutical analysis. Many method issues such as insufficient sensitivity, poor precision, and unusual (too high/low) spiking recovery are often directly related to analytes' instability. We report herein a matrix deactivation approach that chemically stabilizes these analytes for analytical method development. In contrast to the conventional chemical derivatization approach where the analytes are transformed into stable detectable species, the matrix deactivation approach chemically deactivates the hypothetical reactive species in the sample matrix. The matrix deactivation approach was developed on the premise that the instability of certain analytes at trace level is caused by reactions between the analytes and low level reactive species in the sample matrix. Thus, quenching the reactivity of the reactive species would be a key to stabilizing the unstable and reactive analytes. For example, electrophilic alkylators could be destabilized by nucleophiles or bases through either nucleophilic substitution or elimination reactions. One way to mask those reactive species is via protonation by adding acids to the diluent. Alternatively, one can use nucleophile scavengers to deplete reactive unknown species in the sample matrix completely, in analogy to the use of antioxidants and metal chelators to prevent oxidation in the analysis of compounds liable to oxidation. This paper reports the application of the matrix deactivation to the analyses of unstable and reactive pharmaceutical genotoxic impurities. Some of the methods have been used to support development of manufacturing processes for drug substances and a recent regulatory filing.

1. The roles of multidrug resistance-associated protein (Mrp) 2 deficiency and Mrp3 up-regulation were evaluated on the metabolism and disposition of gemfibrozil. 2. Results from in vitro studies in microsomes showed that the hepatic intrinsic clearance (CLint) for the oxidative metabolism of gemfibrozil was slightly higher (1.5-fold) in male TR- rats, which are deficient in Mrp2, than in wild-type Wistar rats, whereas CLint for glucuronidation was similar in both strains. 3. The biliary excretion of intravenously administered [14C]gemfibrozil was significantly impaired in TR-) rats compared with Wistar rats (22 versus 93% of the dose excreted as the acyl glucuronides over 72 h). Additionally, the extent of urinary excretion of radioactivity was much higher in TR- than in Wistar rats (78 versus 2.6% of the dose). 4. There were complex time-dependent changes in the total radioactivity levels and metabolite profiles in plasma, liver and kidney, some of which appeared to be related to the up-regulation of Mrp3. 5. Overall, it was demonstrated that alterations in the expression of the transporters Mrp2 and Mrp3 significantly affected the excretion as well as the secondary metabolism and distribution of [14C]gemfibrozil.

1. Four new metabolites of pioglitazone were identified by liquid chromatography-mass spectrometry (LC-MS/MS) as being formed by hydroxylation (M-VII and M-VIII), opening of the thiazolidinedione ring (M-X) and by desaturation of the terminal ethyl side chain or tether ethoxy moiety (M-IX), respectively. The structure of one of the hydroxylated metabolites (M-VII) was confirmed by chemical modification using the Jones reaction. 2. Oxidative cleavage of the thiazolidinedione ring is a novel pathway not previously reported for pioglitazone. 3. The hydroxylated M-VII was detected in incubations with rat, dog and human liver and kidney microsomes, and in plasma from rats and dogs dosed orally with [(3)H]pioglitazone. 4. The carboxylic acid derivative of M-VII (M-V) and its taurine conjugate were the major radioactive components in dog bile.

An online sample extraction chiral bioanalytical method was developed and validated for the quantification of terbutaline, a β2-selective adrenoceptor agonist, spiked into human plasma by using two extraction columns and a chiral stationary phase (CSP) in conjunction with liquid chromatography tandem mass spectrometry (LC-MS/MS). In this method, two Oasis HLB extraction columns were used in parallel for plasma sample purification and a Chirobiotic T CSP was used for enantiomeric separation. Atmospheric pressure chemical ionization MS/MS was employed in multiple reaction monitoring mode for the detection and quantification. Subsequent to the addition of an internal standard solution, the plasma samples were directly injected onto the system for extraction and analysis. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for reconditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a shorter run time and higher throughput. A lower limit of quantification of 1.0 ng/mL was achieved using only 50 microliter of human plasma. The method was validated with a dynamic range of 1.0–200 ng/mL. The intra- and interday precision was no more than 11% CV and the assay accuracy was between 94–106%. Chirality 14:742–749, 2002. © 2002 Wiley-Liss, Inc.