A sensitive LC-MS-MS assay for the determination of lapatinib in human plasma in subjects with end-stage renal disease receiving hemodialysis

https://doi.org/10.1016/j.jchromb.2018.09.005Get rights and content

Highlights

  • No assays reported for lapatinib analysis for subjects with end-stage renal disease (ESRD) receiving hemodialysis (HD).

  • Subjects with ESRD receiving HD may be treated with medications that may interfere with the quantitation of lapatinib.

  • A LC-MS-MS assay has been validated and used to support a clinical study in subjects with ESRD receiving HD.

Abstract

Most bioanalytical methods reported in literature for the quantitation of lapatinib in human plasma are either for lapatinib alone or lapatinib administered along with other tyrosine kinase inhibitors (TKIs) for therapeutic drug monitoring (TDM) in cancer patients. Recently there was a need for the quantitation of lapatinib in patients with end-stage renal disease (ESRD) receiving hemodialysis (HD). This special patient population normally receives many concomitant medications which have the potential to interfere with the quantitation of lapatinib. Here we describe an LC-MS-MS bioanalytical assay for the quantitation of lapatinib in human plasma containing potential concomitant medications which are commonly given to patients with ESRD receiving HD. The lapatinib calibration curve range was 2.50–1000 ng/mL. Lapatinib was fortified with its isotopically labeled internal standard in a 50 μL plasma aliquot and extracted with protein precipitation. The chromatographic separation was achieved on a Zorbax SB-C18 (5 μm, 2.1 × 50 mm) column with isocratic elution. Assay precision, accuracy, linearity, selectivity, sensitivity and analyte stability covering sample storage and analysis were established. No interferences were observed for the quantitation of lapatinib in the presence of concomitant medications. The validated LC-MS-MS method has been successfully applied to a clinical study for the determination of lapatinib concentrations in human plasma for patients with ESRD receiving HD.

Introduction

Anemia is one of the serious complications in patients with chronic kidney disease (CKD). Anemia is observed in most patients receiving HD and with peritoneal dialysis dependent CKD. Anemia in patients with CKD is a disease state in which the hemoglobin (Hgb) concentration decreases below the normal range in the presence of renal dysfunction. The major cause of anemia in patients with CKD is a decreased ability to produce erythropoietin (EPO) in the kidney associated with renal dysfunction; therefore the production of EPO cannot be increased in response to decreased oxygen concentration in the tissues. Other causes of anemia in patients with CKD are a shortened lifetime of erythrocytes due to uremia caused by reduced renal function, impaired erythropoiesis due to iron and vitamin deficiencies through malnutrition resulting from diet restriction therapy, and loss of residual blood in the circulation after dialysis therapy in patients with HD and peritoneal dialysis dependent CKD [1]. For the treatment of anemia in patients with CKD, erythropoiesis stimulating agents (ESAs) such as recombinant human erythropoietin (rHuEPO) and long-acting EPO are used [2,3]. ESA treatment however, imposes a heavy economic, physical, and mental burden, including pain and risk of infection associated with the injection in patients with CKD. Therefore, new anti-anemia agents that are easier to use and provide better control of Hgb concentrations are needed. JTZ-951 is a potent, orally bioavailable, selective inhibitor of hypoxia inducible factor-Prolyl hydroxylase domain (HIF-PHD). It enhances endogenous EPO production in the kidney and liver leading to enhancement of erythropoiesis which results in increases in hemoglobin concentrations. In vitro transporter studies with human epithelial colorectal adenocarcinoma (Caco-2) cells showed that JTZ-951 is a substrate of breast cancer resistance protein (BCRP). BCRP, an efflux transporter and is highly expressed in human placenta, blood-brain barrier, kidney, small intestine, liver, and other tissues, can affect drug bioavailability, distribution and hepatobiliary clearance. To evaluate the impact of a BCRP inhibitor on the PK profile of JTZ-951, lapatinib, a potent BCRP inhibitor [4], and JTZ-951 were dosed in patients with end-stage renal disease (ESRD) receiving HD in a transporter interaction study. Consequently, a bioanalytical assay in human plasma was developed and validated for the quantitation of lapatinib for this special patient population with ESRD receiving HD.

LC-MS-MS bioanalytical assays for the quantitation of lapatinib have been reported in the literature with various sample preparation procedures which include both on-line and off-line sample extraction procedures. Hsieh et al. reported [5] the first bioanalytical assay in 2004 for the quantitation of lapatinib using 80 μL of human serum with a curve range of 1–1000 ng/mL. Serum samples were extracted by on-line extraction utilizing turbulent flow technique with home-made custom equipment to increase sample throughput. Couchman et al. [6] also used turbulent flow on-line sample extraction with an Aria Transcend TLX-II system for the measurement of lapatinib along with eight other tyrosine kinase inhibitors (TKIs) in human plasma and serum for the purpose of therapeutic drug monitoring (TDM). The assay curve range for lapatinib was 100–5000 ng/mL using a 50-μL sample volume. On-line extraction systems (either homemade or commercially available equipment) may not be readily available to bioanalytical labs and therefore LC-MS-MS assays with off-line sample preparation have also been developed and validated. Off-line sample preparation methods include solid phase extraction (SPE), protein precipitation extraction (PPE), and liquid-liquid extraction (LLE).

For SPE assays, two methods have been reported in the literature. The first off-line SPE sample preparation method coupled with LC-MS-MS for the quantitation of lapatinib in human plasma was reported by Bai et al. in 2006 [7]. The curve range was 100–10,000 ng/mL using a 100 μL sample volume. The assay was used for the quantitation of lapatinib in a Phase I study in children with cancer. Bouchet et al. reported [8] another SPE sample preparation method for the simultaneous analysis of lapatinib and eight other TKIs for TDM in human plasma. This method had better assay sensitivity with a lower limit of quantitation (LLOQ) of 10 ng/mL, but also required a larger sample volume (300 μL of plasma).

Five PPE methods are reported in the literature for the quantitation of lapatinib. The first PPE method for the simultaneous quantitation of lapatinib and five other TKIs in human plasma for TDM was described by Haouala et al. in 2009 [9] with a curve range of 5–5000 ng/mL for lapatinib using a 100 μL sample aliquot. A similar PPE method was reported by Gotze et al. [10] for the measurement of lapatinib and five other TKIs. The sample volume for this assay was also 100 μL but had a higher LLOQ (50 ng/mL) for lapatinib comparing to the method reported by Haouala et al. To reduce the sample volume while maintaining adequate sensitivity for TDM studies, Lankheet et al. [11] reported another PPE assay for the quantitation of lapatinib along with seven other TKIs for support of TDM. The assay used only a 50-μL sample volume to achieve an LLOQ of 20 ng/mL. Andriamanana et al. reported [12] a similar LC-MS-MS method for the quantitation of lapatinib and eight other TKIs. While this method used the same 50 μL sample volume, as that of the method reported by Lankheet et al., the LLOQ (50 ng/mL) was higher than the previous PPE assay from Lankheet's lab. Micova et al. [13] reported a direct injection method without an LC column for the measurement of lapatinib and three other TKIs using PPE as the sample extraction method. This assay achieved an LLOQ of 25 ng/mL for lapatinib using only a 20-μL sample volume.

Three LLE methods are reported in the literature for the measurement of lapatinib. Roche et al. reported [14] the first LC-MS-MS assay for the measurement of cellular levels of lapatinib and dasatinib utilizing LLE for sample extraction. The LLOQ for lapatinib was 31 pg on column. This method was utilized to examine potential mechanisms of pharmacokinetic resistance in cancer cell line models. Musijowski et al. reported [15] a single quadrupole LC-MS method for the quantitation of lapatinib in human plasma. The samples were extracted with LLE and the curve range was 5–800 ng/mL using a 250 μL sample volume. Singhal et al. [16] validated an LC-MS-MS assay utilizing LLE and achieved better sensitivity (LLOQ of 2.5 ng/mL) with less sample volume (100 μL of plasma).

Wu et al. [17] compared the recovery of lapatinib in human plasma using a structure analogue and a stable labeled IS for the quantitation of lapatinib using an LC-MS-MS assay. It is important to note that their results indicated that a stable labeled internal standard was essential for correcting inter-individual variability in the recovery of lapatinib from cancer patient plasma samples.

The methods reported in the literature to date were either for the quantitation of lapatinib alone or for the quantitation of lapatinib along with other TKIs for TDM in cancer patients. Despite the numerous methods, as far as we know, no methods have been reported for the quantitation of lapatinib in subjects with ESRD receiving HD. The subjects with ESRD receiving HD are usually treated with a long list of medications which may interfere with the quantitation of lapatinib. The purpose of this study was to develop and validate an LC-MS-MS assay that could be used to support a clinical study to measure lapatinib in subjects with ESRD receiving HD.

Section snippets

Chemicals and reagents

Reagents and sources used were as follows: Lapatinib (Fig. 1-top) was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada); Lapatanib-d4 (Fig. 1-bottom) from TLC Pharmaceutical Standards (Aurora, Ontario, Canada); HPLC grade acetone from EMD Millipore (Billerica, MA, USA); HPLC grade acetonitrile, methanol, and acetylacetone (≥99%) from Sigma-Aldrich (St. Louis, MO, USA); ammonium formate (≥99.99%) from Aldrich (St. Louis, MO, USA); HPLC grade isopropanol from J.T. Baker

Method development

Chromatographic conditions were optimized by examining various mobile phases, HPLC columns and sample reconstitution solutions to obtain symmetrical peak shape with adequate response, good retention, and minimal carryover. Multiple reversed phase columns were tested during method development. These included the Gemini C18 (2.1 × 50 mm, 3 μm and 5 μm) and the Zorbax SB-C18 (2.1 × 50 mm, 5 μm) columns. The Gemini C18 and Zorbax SB-C18 columns were initially evaluated using 10 mM ammonium formate

Conclusions

A LC-MS-MS bioanalytical method for the determination of lapatinib from a 50-μL human plasma sample aliquot was developed and validated with a dynamic range of 2.50–1000 ng/mL in accordance with FDA guidelines. The validation results demonstrate that the method is simple, sensitive, selective, accurate, precise and reproducible. The method was successfully utilized to support a clinical study in subjects with ESRD receiving HD. The 100% passing rate for all the sample analysis runs and all the

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    Burcu Dogan-Topal et al [14] have examined the electro-oxidation of LAP and its interaction with dsDNA at glassy carbon electrode(GCE) while, Begum Evranos Aksoz et al,[15] have used GCE for the quantification of LAP in the concentration range of 2.0 × 10−8 to 1.0 × 10−6 mol L−1 with a limit of detection(LOD) of 5.71 nM. To the best of our knowledge, only one electroanalytical method [10] is reported for the quantification of LAP at GCE. To develop an electrochemical method for the assay of LAP at the nanomolar level, we have fabricated a simple, sensitive and eco-friendly electrochemical sensing platform based on β-CD@C-dots film on GCE.

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