Development and validation of an assay for a novel ghrelin receptor inverse agonist PF-5190457 and its major hydroxy metabolite (PF-6870961) by LC-MS/MS in human plasma

Abstract

PF-5190457 is a selective and potent ghrelin receptor inverse agonist presently undergoing clinical trials to treat alcohol use disorder (AUD). We describe the development and validation of a selective and sensitive liquid chromatography-tandem mass spectrometry-based method for quantification of PF-5190457 and its recently discovered hydroxy metabolite PF-6870961 in human plasma. Analytes were extracted after simple protein precipitation using methanol (2.5 ng mL⁻¹ tacrine as an internal standard). A gradient liquid chromatography method was used to separate the analytes on an Acquity UPLC BEH C18 analytical column. The separation was achieved at a flow rate of 0.25 mL min⁻¹ and the total chromatographic runtime was 11.30 min. Positive electrospray ionization and multiple reaction monitoring mode were used for the quantification of all the analytes. The calibration curves from six validation runs were linear with a correlation coefficient of ≥0.996 for the concentration range of 1–1000 ng mL⁻¹ and 2–250 ng mL⁻¹ for PF-5190457 and PF-6870961, respectively. The retention time for PF-5190457, PF-6870961 and tacrine were 4.4, 3.8, and 4.6 min, respectively. The lower limit of quantification for PF-5190457 and PF-6870961 was 1 and 2 ng mL⁻¹, respectively. The inter-assay precision and accuracy results obtained were within the Food and Drug Administration recommended ± 15% limit of nominal values. All the analytes were found to be stable under varied stability conditions. The recovery of PF-5190457 and PF-6870961 ranged from 95 to 103%. Further, the application of the method was demonstrated by measuring the concentration of PF-5190457 and its hydroxy metabolite in patient plasma samples from 100 mg dose.

1. Introduction

Alcohol consumption is frequent in the United States with a significant increase in the prevalence of alcohol use in the last 10 years [1]. From a clinical standpoint, AUD is characterized by distress, compulsive alcohol intake, and inability to control alcohol use [2]. The US economic burden of alcohol misuse and associated diseases is estimated to be around $250 billion [3]. Heavy alcohol consumption may also accelerate the progression of scarring and fibrosis in patients with NAFLD leading to the risk of hepatocellular carcinoma [4]. Patients with alcoholic and non-alcoholic fatty liver have also been associated with alteration of drug metabolizing enzymes in human liver [5,6]. Recent decade has seen a continuous research effort for discovery and development of therapeutic agents with the potential to ameliorate AUD [7,8].

PF-5190457, a novel peripherally acting inverse agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), also known as ghrelin receptor, and a member of spiro-azetidine-piperidine series, was discovered by Pfizer Inc. [9,10]. Ghrelin, a peptide released by endocrine cells localized primarily in the stomach, is the endogenous ligand for GHS-R1a and regulates appetite, meal initiation, and food intake [11–13]. Antagonists of the ghrelin receptor have been explored for their therapeutic potential in the treatment of type 2 diabetes mellitus, eating disorders, obesity, and alcohol use disorder. Such antagonists have the potential to stimulate glucose-dependent insulin secretion, reduce food intake, and reduce the craving for alcohol consumption [14–20]. PF-5190457 is currently undergoing clinical trials for the treatment for alcohol use disorder [16,21,22].

Our lab has previously reported the validation of a UPLC-MS/MS method for PF-5190457 [21]. However, a primary metabolite, PF-6870961 of the drug was recently discovered in human plasma [23]. Here, we describe an ultra-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) assay for simultaneous determination of PF-5190457 and its hydroxy metabolite (PF-6870961) in human plasma using tacrine as an internal standard (IS).

2. Materials and methods

2.1. Chemicals and reagents

PF-5190457 and tacrine were obtained from Sigma-Aldrich (St. Louis, MO). PF-6870961 (hydroxy metabolite) was provided by Pfizer Inc. under a confidentiality agreement with The University of Rhode Island (Pfizer Inc., Groton, CT). LC-MS grade methanol and formic acid were obtained from Fisher Scientific (Fair Lawn, NJ). Blank K₂EDTA human plasma from six subjects (three males, three females) was obtained from Bioreclamation IVT (Westbury, NY).

2.2. LC-MS/MS analysis

LC-MS/MS analysis was performed on an Acquity UPLC^® system coupled with Xevo^® TQ MS detector (Waters Corp, Milford, MA, USA). MassLynx^™ Software (V 4.1) and TargetLynx^™ (V 4.1) were used for data processing. Chromatography separation of analytes and IS was accomplished with a run time of 11.30 min using a gradient elution method. Analytes were resolved on an Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm particle size, 130Å pore size) analytical column equipped with an Acquity UPLC BEH C18 VanGuard pre-column (2.1 × 5 mm, 130 Å). Mobile phase consisted of water: methanol 95:5% (v/v) with 0.1% formic acid (A) and 100% methanol with 0.1% formic acid (B). The column and autosampler temperature were kept at 40 °C and 10 °C, respectively. The mobile phase was delivered at 0.25 mL min⁻¹ flow rate, and the injection volume was 5 μL. The initial conditions of the mobile phase (98% A, 2% B) were maintained for 2 min, B was increased from 2% to 80% over 8.30 min, and held till 9.30 min before being returned to initial conditions, followed by a 2 min equilibration for a total run time of 11.30 min.

Mass spectral analysis and quantification were carried out using multiple reaction monitoring (MRM) in positive electrospray ionization mode with the following parameters: desolvation temperature 350 °C, desolvation gas flow 650 L h⁻¹, capillary voltage 3.50 kV and cone voltage 20 V. The fragmentation pattern for PF-5190457 and IS were similar to previously reported methods [21,24,25]. The optimized settings of mass spectrometer voltage, precursor and fragment m/z and the retention time for analytes and internal standard are summarized in Table 1.

Table 1.

Mass spectrometry parameters for analytes and internal standard.

Compounds	Precursor (m/z)	Fragment (m/z)	Dwell time (s)	Cone voltage (V)	Collision energy (V)	Retention time (min)
PF-5190457	513	209, 335	0.025	30, 30	44, 16	4.4
Hydroxy metabolite (PF-6870961)	529	225, 351	0.025	34, 32	40, 20	3.8
Tacrine	199	170, 143	0.025	46, 42	30, 36	4.6

2.3. Preparation of solutions

Sub-stock solutions of PF-5190457 and PF-6870961 were separately prepared in 50% methanol to obtain a final concentration of 10 μg/mL and were stored at 4 °C. Working stock solutions for calibration and quality controls were prepared from the stock solution by adequate dilution using diluent (methanol: water, 50:50, v/v). The calibration standards and quality control samples were prepared by spiking an appropriate amount of the standard working solutions into pooled K₂EDTA human plasma with an organic solvent concentration < 5% of the final volume. An eight-point calibration curve with concentrations ranging from 1–1000 ng mL⁻¹ and 2–250 ng mL⁻¹ for PF-5190457 and PF-6870961 were prepared respectively. Low, middle, and high-level QC samples were prepared for PF-5190457 (3, 200 and 750 ng/ml) and PF-6870961 (5, 25 and 200 ng/ml). A final working solution of 2.5 ng/mL tacrine (IS) in methanol was used as a precipitating solution.

2.4. Extraction of analytes from plasma

Samples were prepared by a simple protein precipitation method. Two-hundred μL of precipitating solution containing IS was added to 50 μL of plasma. Samples spiked with analytes were extracted with methanol containing IS whereas, the double blank was extracted in 100% methanol without any IS. The mixture was vortex mixed for 15 s and further centrifuged at 10,000 rpm for 5 min at 10 °C. The supernatant was collected and used for further analysis using LC-MS/MS method as described above.

2.5. Method validation

The validation of this assay was performed according to the requirements as outlined in guidelines established by the United States Food and Drug Administration (US FDA). The method was evaluated for selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect, recovery, and stability of analytes [26].

2.5.1. Specificity and sensitivity

The method specificity was evaluated before the main validation batches for the presence of interference from endogenous compounds by screening blank plasma of six donors. The sensitivity of the assay was evaluated by analyzing blank plasma samples from six different donors and spiked plasma samples at the lower limit of quantification (LLOQ) levels of the analytes.

2.5.2. Linearity, accuracy, and precision

The linearity of the method was determined by analysis of standard plots associated with an eight-point calibration curve. Inter-assay precision and accuracy were calculated from the replicates in six different analytical runs. Peak area ratios of analytes to IS obtained from MRM were utilized for the construction of calibration curves, using least squares weighted (1/x) linear regression. Autosampler carry-over test was assessed to demonstrate any carryover of analytes and IS.

2.5.3. Matrix effect and recovery

The components present in the matrix (i.e., extracted from plasma) can interfere with ionization efficiency by coeluting with analytes of interest [27]. Matrix effect for ion suppression and enhancement was assessed using blank and spiked human plasma using post-column infusion experiment as described previously [21,28]. The chromatograms were visually evaluated to verify for any interfering peaks at the elution time of analytes and IS. Likewise, the interference of phospholipids was determined since these are highly abundant endogenous compounds in biological membranes and can interfere with the elution of analytes [29,30]. Phospholipids were monitored by measuring the precursor ion (Q1) m/z 496, 522, 524, 758 and 782 and their product ion (Q3) m/z 184 [31,32]. Recovery was determined at two concentration levels (LQC and HQC) by comparing the peak area of the analytes from the pre-extracted plasma standard with that obtained from the post-extracted standard at the same concentration for the QC samples.

2.5.4. Stability

Stability of analytes was evaluated at two concentrations (LQC and HQC). Freeze-thaw stability (three cycles), benchtop stability (6 h on lab-bench), and short-term stability (1-week at −80 °C) were assessed as a part of method validation. The autosampler storage stability was determined by storing the QC samples for ~48 h under autosampler condition (10 °C) before being analyzed.

2.5.5. Method application

The results from the previously validated UPLC-MS/MS method for the determination of PF-5190457 in patient plasma samples (n = 45) were correlated with the current method to test the assay quality of current method [21]. We used plasma samples collected at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 22.5 and 24 h after oral administration of 100 mg PF-5190457 from a phase 1b clinical study with heavy drinkers who also had a diagnosis (except one) of alcohol dependence. The study was conducted at the National Institutes of Health Clinical Center (Bethesda, MD, USA). All required regulatory approvals were obtained, and all participants signed a written consent document before research procedures began (NCT02039349). The main results of this clinical study have been reported elsewhere; please see Lee et al. [22]. Incurred sample reanalysis (ISR) was performed to corroborate the results utilizing clinical samples. Samples (10% of the study size) around Cmax and terminal elimination phase were selected, reanalyzed, and compared to the initial results obtained from clinical samples (described below). Samples were extracted as described above. The results of the repeated analysis were compared with original data, according to the following formula: (repeated sample − original sample) × 100/mean value. Two-thirds of the samples reanalyzed should be within ± 20% between the two analyses [33].

3. Results and discussion

3.1. LC-MS/MS assay performance

The goal of this work was to develop and validate a sensitive and straightforward assay method for the simultaneous determination of PF-5190457 and its hydroxy metabolite (PF-6870961) in human plasma. The MS optimization was performed by direct infusion of solutions of PF-5190457, PF-6870961, and IS into the ESI source of the mass spectrometer. The best signal for both the analytes and IS was achieved using a positive ion mode. Protonated adducts for PF-5190457 (m/z 513) and PF-6870961 (m/z 529) and their respective high-intensity fragments (m/z 209 and m/z 225, respectively) were selected for quantification.

Several iterations of chromatographic conditions were performed to select an optimal mobile phase and analytical C18 columns. As the metabolite was found to be unstable in acetonitrile, we used methanol as an organic additive to separate the metabolites in this method. While the previously published method for quantification of parent had a run time of 2.2 min, we found difficulties in adequate separation of metabolites using a shorter method. During subsequent trials, we found that a longer method provided good separation of major metabolite from the minor metabolites observed in human plasma. These trials determined that the present method achieve an optimal degree of chromatographic separation for simultaneous separation of the major metabolite as well as the parent drug.

3.2. Sample preparation optimization

A simple protein precipitation technique was utilized for the extraction of analytes from the plasma samples. Acetonitrile and methanol were investigated for the extraction of analytes from plasma. We found that the PF-5190457 metabolites were unstable in acetonitrile. Therefore, methanol was chosen as the organic solvent for the preparation of the stock solution for the analytes and IS as well as only methanol was used in the mobile phase.

3.3. Selection of internal standard

The stable isotopic labeled IS was not available for the hydroxy metabolite, and therefore we have demonstrated the suitability of readily available, cost-effective compounds with no evidence of crosstalk with the analytes of interest in the current assay. Various compounds with similar logP and pKa to that of analytes including indoprofen, chlorzoxazone, naproxen, tacrine, and rifampicin were tested for selection of suitable IS for the assay (data not shown). We used tacrine after initial experiments where we found suppression of the parent and deuterated IS as the concentration of metabolite increased. We found that tacrine gave a stable signal which was not suppressed by any of the analytes and used it for subsequent method validation. Additionally, tacrine ionizes in the positive electrospray and elutes in the vicinity of the analytes.

3.4. Method validation

3.4.1. Specificity and selectivity

The determination of PF-5190457, PF-6870961, and IS at their respective retention time were not affected by interference from co-extracted matrix components under the present LC-MS/MS conditions. There was no interference observed from endogenous compounds in blank plasma obtained from six different donors. The representative extracted ion chromatograms of the analytes and the IS in human plasma are given in Fig. 1(a–c).

Fig. 1.

Representative chromatograms of (a) plasma sample extracted with IS (b) plasma sample spiked at the lower limit of quantification for PF-5190457 and its hydroxy metabolite and extracted with 2.5 ng/mL of tacrine and (c) drug-free plasma sample.

3.4.2. Linearity, precision, and accuracy

Calibration was found to be linear over the concentration range of 1–1000 ng mL⁻¹ for PF-5190457 and 2–250 ng ml⁻¹ for PF-6870961. The curve was fitted with linear regression using 1/x weighing function (Table 2). The correlation coefficients (r²) based on linear regression for both the analytes was > 0.996. Precision and accuracy for this method were controlled by calculating the inter-batch variations of LLOQ and QC samples at four concentrations for PF-5190457 (1, 3, 200 and 750 ng/mL) and PF-6870961 (2, 5, 25 and 200 ng/mL) as shown in Table 3. The inter-day precision of 2.4–7.4% for PF-5190457 and 3.6–6% for PF-6870961. This method demonstrated inter-day accuracy 88.0–104.2% for PF-5190457 and 97.2–109.7% for PF-6870961. There was no between-sample carryover detected following the double blank injections.

Table 2.

Summary of calibration standards from six validation runs.

Analyte		STD 1	STD 2	STD 3	STD 4	STD 5	STD 6	STD 7	STD 8	r2
PF-5190457	Nominal conc. (ng mL−1)	1	2	10	100	250	500	800	1000
	Mean conc. (ng mL−1)	1.0	1.9	11.2	108.8	266.8	521.2	822.3	936.4	0.99
	SD	0.0	0.2	0.4	3.8	8.4	10.2	20.5	20.3
	%Bias	0.9	−4.8	12.0	8.8	6.7	4.2	2.8	−6.4
	CV	3.9	8.2	3.7	3.5	3.2	2.0	2.5	2.2
	%Accuracy	99.1	104.8	88.0	91.2	93.3	95.8	97.2	106.4
Analyte		STD 1	STD 2	STD 3	STD 4	STD 5	STD 6	STD 7	STD 8	r2
Hydroxy metabolite (PF-6870961)	Nominal conc. (ng mL−1)	2.0	3.9	7.8	15.6	31.5	62.5	125.0	250.0
	Mean conc. (ng mL−1)	2.1	3.4	6.9	13.5	28.3	58.4	127.6	256.7	0.99
	SD	0.1	0.1	0.8	0.8	0.9	3.4	1.6	2.4
	%Bias	3.4	−12.9	−11.7	−13.2	−10.1	−6.6	2.1	2.7
	%CV	5.8	2.5	12.2	5.8	3.0	5.7	1.2	0.9
	%Accuracy	96.6	112.9	111.7	111.6	110.1	106.6	97.9	97.3

n = 6 (one replicate for each of the six validation runs), %Accuracy = 100 − [(mean − nominal)/nominal] * 100, %CV calculated as (SD/mean) * 100, % bias = 100 × (mean − nominal)/nominal.

Table 3.

Precision and accuracy analysis from six validation runs.

		LLOQ	LQC	MQC	HQC
PF-5190457	Nominal conc. (ng/mL−1)	1.0	3.0	200.0	750.0
	Inter-run mean	1.0	2.9	224.0	776.6
	Inter-run SD	0.1	0.2	5.3	28.3
	Inter-run %bias	4.9	−4.2	12.0	3.5
	Inter-run CV	11.4	7.6	2.4	3.6
	% Accuracy	95.1	104.2	88.0	96.5
		LLOQ	LQC	MQC	HQC
Hydroxy metabolite (PF-6870961)	Nominal conc. (ng/mL−1)	2.0	5.0	25.0	200.0
	Inter-run mean	2.0	4.9	22.6	205.5
	Inter-run SD	0.1	0.2	0.8	9.8
	Inter-run %bias	2.1	−2.6	−9.7	2.8
	Inter-run CV	6.0	4.7	3.6	4.8
	% Accuracy	97.9	102.6	109.7	97.2

n = 18 (three replicates for each of the six validation runs), %Accuracy = 100 − [(mean − nominal)/nominal] * 100, %CV calculated as (SD/mean) * 100, %bias = 100 × (mean − nominal)/nominal.

3.4.3. Matrix effect and recovery

The determination of PF-5190457 and PF-6870961 was not affected by any co-extracted matrix components under the LC-MS/MS conditions used (Supplementary Fig. 1). There was no interference from the phospholipids at a retention time of elution of analytes (Supplementary Fig. 2). The percent mean recovery for PF-5190457 and PF-6870961 ranged from 95 to 103%.

3.4.4. Stability

The data representing the stability of analytes in plasma at two concentration levels (LQC and HQC) is given in Table 4. These results indicate that the analytes are stable in human plasma as the mean of the results of the tested samples were within the acceptance criteria of ± 15% of the nominal concentrations. These findings suggest no stability-related problems would be expected during usual circumstances of a pharmacokinetic sample collection and quantification of drug or metabolite concentrations.

Table 4.

Summary of stability studies.

		Bench top (6 h)		Freeze thaw (3 cycles)		Autosampler (48 h)		Short-term (1 week)
		LQC	HQC	LQC	HQC	LQC	HQC	LQC	HQC
PF-5190457	Nominal conc. (ng/mL−1)	3.0	750	3.0	750	3.0	750	3.0	750
	Mean conc. (ng/mL−1)	3.0	795.0	2.7	782.2	2.8	759.1	3.0	759.3
	% Bias	0.3	6.0	−9.4	4.3	−5.3	1.2	−0.8	1.2
	% CV	9.6	3.7	6.4	4.2	2.2	3.1	2.1	3.1
Hydroxy Metabolite (PF-6870961)	Nominal conc. (ng/mL−1)	5.0	200	5.0	200	5.0	200	5.0	200
	Mean conc. (ng/mL−1)	5.0	215.2	4.8	222.3	−1.5	211.0	4.2	220.0
	% Bias	−3.9	7.6	−5.9	11.1	−3.3	5.5	1.0	10.0
	% CV	7.7	2.8	7.0	0.9	8.2	3.8	4.6	0.8

%CV calculated as (SD/mean) * 100, %bias = 100 × (mean − nominal)/nominal.

LQC and HQC for PF-5190547 and hydroxy metabolite (PF-6870961) are 3 and 750 ng/mL, 5 and 200 ng/mL respectively.

3.4.5. Analysis of clinical samples

Clinical samples (n = 45) previously analyzed by UPLC-MS/MS method and subsequently analyzed by the current method were within ± 15.2% for PF-5190457 (Fig. 2a , and band ). The data generated by this method is reproducible despite differing the LC-MS/MS and sample preparation conditions. Method robustness and reproducibility was also determined by ISR (n = 45) following the FDA Guidance for Industry for parent and metabolite. More than two-thirds of the samples were within ± 20%. The incurred sample analysis also indicated that both parent and its metabolite were stable within human plasma from subjects during storage. Bland-Altman plot is recommended over Pearson’s or Spearman’s correlation coefficient for correlation analysis for ISA as the range of data can influence the correlation [33]. The correlation between both the LC-MS/MS methods for PF-5190457 and PF-6870961 are depicted via Bland–Altman plots in Supplementary Fig. 3.

Fig. 2.

Bland–Altman plots for ISR (Incurred sample reanalysis) showing the correlation for original vs. repeat analysis for (a) PF-5190457 (lower = −2.7%, upper = 22.7%) and (b) PF-687hydroxy metabolite (lower = −22%, upper = 11%) respectively.

3.5. Method application

The suitability of the developed method was evaluated by quantifying the analyte concentration in the plasma from a previously conducted study [22]. The plasma concentration ranges of PF-5190457 and PF-6870961 were measurable within their calibration curves. Fig. 3 represents the plasma concentration-time profile of PF-5190457 and PF-6870961 in a representative study subject after 100 mg oral dose. It was interesting to find that a significant amount of parent (PF-5190457) was metabolized to its hydroxy metabolite (PF-6870961). We recently reported the role of molybdenum containing enzymes in the generation of major hydroxy-metabolite of PF-5190457. While these findings are preliminary, further investigation on pharmacological effect of the metabolite is warranted. Future clinical pharmacokinetic studies would benefit from this validated method for quantification of both the parent and major metabolite in a single LC-MS/MS method.

Fig. 3.

Plasma concentration-time profile of PF-5190457 and its major hydroxy metabolite after an oral dose of 100 mg dose in adult human subject.

4. Conclusion

To conclude, we describe the development of a sensitive LC-MS/MS method for the accurate, precise, and reliable measurement of PF-5190457 and its hydroxy metabolite, PF-6870961 in human plasma. The method also demonstrated the suitability of tacrine as IS for accurate quantification of PF-5190457 and PF-6870961. This method demonstrated adequate reliability and reproducibility of the results within the tested analytical range. This assay was successfully employed for determining the concentration of PF-5190457 and its major metabolite PF-6870961 in plasma sample collected during a phase 1b clinical trial. Given the potential role of PF-5190457 as a novel treatment for alcohol use disorder and more, in general, the potential role of the ghrelin system as a novel target towards the development of novel treatments for alcohol and substance use disorders, the method can be used for quantification of metabolites and drug in future clinical studies [34–37].

Supplementary Material

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jchromb.2019.121820.

Abbreviations:

CV

coefficient of variation

ESI

electrospray ionization

FA

formic acid

IS

internal standard

LLOQ

lower limit of quantification

LQC

lower quality control

ME

matrix effect

MeOH

methanol

MQC

middle quality control

MRM

multiple reaction monitoring

MS

mass spectrometry

MW

molecular weight

PLs

phospholipids

QCs

quality controls

SD

standard deviation

UPLC-MS/MS

ultra-performance liquid chromatography-tandem mass spectrometry