Development and validation of an UPLC–MS/MS method for the quantification of irinotecan, SN-38 and SN-38 glucuronide in plasma, urine, feces, liver and kidney: Application to a pharmacokinetic study of irinotecan in rats

Highlights

• Developed a UPLC-MS/MS method to simultaneously quantitate irinotecan and its metabolites in different bio-matrices and metabolic organs.

• Validated the sensitive and robust LCMS method with respect to precision and accuracy in different bio-matrices and organs.

• Showed in comparison to SN-38 and its glucuronidated form, irinotecan excreted predominantly as unchanged form through urine.

• Found the fecal excretion of SN-38 glucuronide is negligible with respect to irinotecan and SN-38.

Abstract

The objective of this research is to develop and validate a sensitive and reproducible UPLC–MS/MS method to quantify irinotecan, its active metabolite SN-38 and SN-38 glucuronide (phase II metabolite of SN-38) simultaneously in different bio-matrices (plasma, urine, feces), tissues (liver and kidney) and to use the method to investigate its pharmacokinetic behavior in rats. Irinotecan, SN-38 and SN-38 glucuronide has been resolved and separated by C18 column using acetonitrile and 0.1% formic acid in water used as the mobile phases. Triple quadruple mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode were employed to perform mass analysis. The results showed that the linear response range of irinotecan and SN-38 in plasma, feces, liver and kidney is 4.88–10000 nM, 39–5000 nM, 48.8–6250 nM and 48.8–6250 nM, respectively (R² > 0.99). In case of SN-38 glucuronide, the standard curves were linear in the concentration range of 6.25–2000 nM, 4.88–1250 nM, 9.8–1250 nM and 9.8–1250 nM in plasma, feces, liver and kidney homogenates, respectively. The lower limit of detection (LLOD) of irinotecan, SN-38 and SN-38 glucuronide was determined to be less than 25 nM in all bio-matrices as well as tissue homogenates. Recoveries of irinotecan, SN-38 and SN-38 glucuronide at three different concentrations (low, medium and high) were not less than 85% at three different concentrations in plasma and feces. The percentage matrix factors in different bio-matrices and tissues were within 20%. The UPLC–MS/MS method was validated with intra-day and inter-day precision of less than 15% in plasma, feces, liver and kidney. Owing to the high sensitivity of this method, only 20 μl of plasma, urine and homogenates of liver, kidney and feces is needed. The validated method has been successfully employed for pharmacokinetic evaluation of irinotecan in male wistar rats to quantify irinotecan, SN-38 and SN-38 glucuronide in plasma, feces, and urine samples.

Introduction

Irinotecan (7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecine, CPT-11, Fig. 1B) is an important component of FOLFIRI and FOLFIRINOX chemotherapy regimen which are used as first line treatment against colorectal and advanced pancreatic cancer [1], [2]. It has also shown clinical activity against other types of cancers such as lung, ovarian, cervical, gastric, refractory lymphoma and leukemia etc. [3], [4], [5]. Irinotecan is a pentacyclic semisynthetic derivative of camptothecin which is isolated from the bark of a tree named Camptotheca acuminate in 1966 [6], [7]. Irinotecan, through the action of liver carboxylesterase gets converted into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin, Fig. 1C) which is 100–1000 times as potent as irinotecan [8], [9]. Anticancer activity of irinotecan is attributed to the inhibitory effect of SN-38 on DNA topoisomerase I which plays an important role in DNA replication and transcription [10], [11]. Formation of an irreversible complex of SN-38, DNA topoisomerase I enzyme and the ligated DNA strand leads to the breakage of double stranded DNA which ultimately causes cell death. Owing to the poor solubility of SN-38, irinotecan is currently used as a prodrug in clinical applications.

Irinotecan exhibits a complicated metabolic profile by interacting with various drug metabolizing enzymes as well as efflux transporters. Studies indicated that both biliary as well as intestinal excretion of irinotecan and its metabolites are regarded as significant contributing factors to elevate the colonic SN-38 content which causes late onset diarrhea [12], [13], [14], [15], [16]. In spite of knowing the fact that predominant efflux transporters (P-gp and MRP2) mediate the excretion of irinotecan and its metabolites, the role of efflux transporters on the disposition of irinotecan and its metabolites at different metabolic organs (liver, intestine and kidney) has not been clearly defined till date. Quantification of irinotecan and its metabolites in different bio-matrices (bile, plasma, intestinal perfusate and urine) as well as in different metabolic organs (liver and kidney) will allow us to elucidate the role of predominant efflux transporters on the disposition and the intestinal toxicity of irinotecan.

Generally, inactivation of SN-38 to its phase II metabolite SN-38 glucuronide (Fig. 1D) occurs through an enzymatic reaction mediated by the UDP-glucuronosyltransferase 1A1 isoform (UGT1A1) [17], [18]. UGT1A1 plays an important role in the disposition as well as in the intestinal toxicity of irinotecan. In the colon, SN-38 glucuronide, after interacting with the bacterial β glucuronidase enzyme secreted from commensal microbiota, can regenerate toxic SN-38 which has the potential to kill the normal cells in the intestine resulting in late onset diarrhea [19]. At present, several compounds are being synthesized which can predominantly inhibit the activity of the bacterial β-glucuronidase resulting in the reduction of the generation of SN-38 from inactive SN-38 glucuronide [20]. In addition, preclinical studies indicated that around 2–22% of SN-38 glucuronide was excreted into the bile over 24 h which was comparatively higher than biliary excretion of SN-38 (7–9%) [15], [21]. Similarly clinical study of irinotecan also indicated that around 3% of dose is excreted through urine [12]. So in this regard, direct quantification of SN-38 glucuronide in different biomatrices as well as in major metabolic organs will enable us to get the holistic idea of the disposition of irinotecan.

It has been shown that the intact lactone ring of camptothecin (CPT) derivative is an important factor to retain its anticancer activity [10], [11], [22]. However, the lactone ring of CPT derivatives undergoes a pH sensitive, reversible hydrolysis and converts into carboxylate form at physiologic pH [23]. Boyd et al. showed that pH played an important role in the inter conversion of lactone and carboxylate forms of CPT in the solution. Experiments showed that at pH 3–5, lactone is the most stable form of CPT, whereas at pH 9, it rapidly converts into carboxylate form [24]. Till date, HPLC methods coupled with fluorescence detectors have been the most predominant method for detection of CPT derivatives as they can detect both lactone and carboxylate forms in different biological matrices.

Different bioanalytical techniques, mostly based on reverse phase liquid chromatography coupled with fluorescence detection, have been proposed to determine irinotecan and SN-38 in various biological matrices. However, these fluorescence methods suffered from several drawbacks such as lengthy preparation time, limited sensitivity and high sample volumes [25], [26], [27]. Recently, liquid chromatography coupled with mass spectrometry (LC–MS and LC–MS/MS) has become one of the preferred analytical tools for the rapid and efficient quantification of small and large molecules in different biological matrices due to the unique combination of high specificity, sensitivity and high sample throughput possibilities. Although several LC–MS/MS methods have been developed and implemented for the quantification of irinotecan and SN-38 in rabbit, mouse and human plasma, very few of them developed a UPLC–MS/MS method of SN-38 glucuronide [28], [29], [30], [31]. Similarly, though quantification of irinotecan and SN-38 was done in mouse tissues (brain, liver, kidney etc.), there was no report of the quantification of SN-38 glucuronide in these tissues [32], [33]. Also, to the best of our knowledge there is no LC–MS/MS method available to quantify irinotecan, SN-38 and SN-38 glucuronide simultaneously in urine and feces. In this study, we developed and validated a simple, rapid and sensitive LC–MS/MS method for simultaneous quantification of total concentration of irinotecan, SN-38 and SN-38 glucuronide in rat plasma, feces, urine, liver and kidney homogenate to support pharmacokinetic studies in rats.