A positive-negative switching LC-MS/MS method for quantification of fenoldopam and its phase II metabolites: Applications to a pharmacokinetic study in rats

Abstract

Fenoldopam is an approved drug used to treat hypotension. The purpose of this study is to develop and to validate an LC-MS method to quantify fenoldopam and its major metabolites fenoldopam-glucuronide and fenoldopam-sulfate in plasma and to apply the method to a pharmacokinetic study in rats. A Waters C₁₈ column was used with 0.1% formic acid in acetonitrile and 0.1% formic acid in water as the mobile phases to elute the analytes. A positive-negative switching method was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM). A one-step protein precipitation using methanol and ethyl acetate was successfully applied for plasma sample preparation. The method was validated following the FDA guidance. The results show that the LLOQ of fenoldopam, fenoldopam-glucuronide and fenoldopam-sulfate is 0.98, 9.75 and 0.98 nM, respectively. The intraday and interday variance is less than 8.4 % and the accuracy is between 82.5–116.0 %. The extraction recovery for these three analytes ranged from 81.3 ± 4.1% to 113.9 ± 13.2%. There was no significant matrix effect and no significant degradation under experimental conditions. PK studies showed that fenoldopam was rapidly eliminated (t_1/2= 0.63 ± 0.24 h) from the plasma and glucuronide is the major metabolite. These methods were suitably selective and sensitive for pharmacokinetic and phase II metabolism studies.

1. Introduction

Fenoldopam is a selective dopamine 1 receptor agonist which causes peripheral vasodilation. Pharmacological studies showed that low doses of fenoldopam increase renal blood flow without affecting blood pressure, while high doses of fenoldopam can reduce blood pressure and maintain renal perfusion[1]. The drug was approved to treat hypertensive urgencies in 1997 through the IV route of administration. Other than the approved indication, fenoldopam has also been frequently tested in humans in treating certain kidney diseases, such as early acute kidney injury after a liver transplant [2], oliguria/anuria and for renal perfusion and protection[3, 4], and renal dysfunction[5], due to its vasodilative function on renal arteries. Fenoldopam has also been reported to have the potential to treat psoriasis due to its anti-proliferative activity[6].

Preclinical and clinical pharmacokinetic studies showed that the clearance of fenoldopam is very rapid[7, 8]. For example, a clinical study demonstrated that the terminal half-life (t1/2) of fenoldopam was only 7.52 ± 1.23 min and plasma drug concentration decreased rapidly after infusion was terminated [7, 8]. This rapid clearance of fenoldopam is probably due to phase 2 metabolism such as glucuronidation and sulfonation because there are three hydroxyl moieties in its structure (Fig. 1), which renders fenoldopam a good substrate for phase II metabolic enzymes (e.g., UGTs and SULTs). Also, in vitro studies showed that glucuronides and sulfates can be formed when fenoldopam was incubated with human liver microsomes [9]. Moreover, fenoldopam is very potent and the typical clinical dose is at μg/kg/min (e.g., 0.5–2 μg/kg/min) range. Therefore, it is critical to monitor the exposure of fenoldopam and its metabolites in the plasma.

Figure 1.

The typical chromatograms of fenoldopam, fenoldopam-glucuronide, fenoldopam-sulfate and internal standard in LC-MS.

The importance of monitoring plasma exposure of fenoldopam has been noticed and different analytical methods have been developed. The earliest reported method was developed in late 1980s using a HPLC with electrochemical detection (CE), where a large volume of plasma was required due to relatively low detection limit [10]. Later, high-performance liquid chromatography with electrochemical detection (HPLC-ED) after ethyl acetate extraction from plasma or urine was developed for the determination of fenoldopam and its identified metabolites in biological matrix. However, the method could not quantify metabolites and hydrolysis has to be conducted to release fenoldopam before analysis. To determine fenoldopam and its metabolites, a single glassy carbon electrode (for fenoldopam) or dual glassy carbon electrodes (for 8-SO4 and methoxy metabolites) were used and developed with a single extraction procedure. However, samples have to be measured twice because fenoldopam and its metabolite have to be determined using different electrodes [9–12]. Thereafter, LC-MS methods were developed in human studies[8, 13], but only for the parent compound--fenoldopam. Therefore, a simple, rapid and direct method to simultaneously quantify fenoldopam and its metabolites is needed.

In this study, we developed a sensitive, rapid and reproducible UHPLC-MS/MS method to simultaneously quantify fenoldopam and its major metabolites fenoldopam-glucuronide and fenoldopam-sulfate in plasma using one step protein precipitation, and to apply the method in a PK study in rats. A method to quantify both parent compound and major metabolites is particularly important for fenoldopam to achieve a more accurate drug in vivo exposure evaluation because the parent compound is rapidly metabolized in vivo.

2. Experimental

2.1. Chemicals and reagents.

Fenoldopam mesylate was purchased from Toronto Research Chemicals (Toronto, Canada, all compounds purity was ≥99%). Uridine-5’-diphosphate-β,D-gluconic acid ester (UDPGA), 3’-phosphoadenosine-5’-phosphosulfate (PAPS), β-glucuronidase, sulfatase, and formononetin (internal standard, IS) were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile, methanol, and water (LC-MS grade) were purchased from EMD (Gibbstown NJ). All other materials (typically analytical grade) were used as received.

2.2. Instruments and conditions

2.2.1. UHPLC system for LC-MS analysis:

System was Exion LC analytical UHPLC systems (SCIEX company, CA, USA). The column was Acquity UPLC BEH C18 column (50 × 2.1 mm I.D. 1.7 μM, Waters, Milford, MA, USA). The 0.1% formic acid was used as mobile phase A and acetonitrile was used as mobile phase B. The gradients were 0 to 0.5 min, 0 to 5 % B, 0.5 to 2.0 min, 5 to 30% B, 2.0 to 3.0 min, 30–70 % B, 3.0 to 4.0 min, 70 to 95 % B, 4.0 to 4.5 min, 95% B, 4.5 to 5 min, 95–100%, 5 to 5.5 min, 100%B; flow rate was 0.4 ml/min, and the column temperature was 40°C. The injection volume was 5 μL and autosampler temperature was 4 °C.

UHPLC-UV system for analysis for metabolite identification: System was Waters Acquity™ UPLC system with a PDA detector (Waters, Milford, MA, USA). Other conditions (i.e., column, mobile phase, gradient, temperatures, injection volume, flow rates) were identical with those described in the UHPLC-MS analysis.

2.2.2. MS system:

MS analysis was performed using an API 5500 Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDS SCIEX, Foster City, CA, USA) equipped with a TurboIonSpray™. The concentrations of fenoldopam and its metabolites were determined by using the multiple reactions monitoring (MRM) scan type with Positive-negative switching to monitor the analyte. Unit mass resolution was set in both mass-resolving quadruple Q1 and Q3 in positive and negative scan mode. ESI Source parameters are: positive ion spray voltage was 5000 V and negative spray voltage was −4500 V; ion source temperature was 450°C, curtain gas flow: 30 psi; nebulizer gas (gas 1), nitrogen, 30 psi; turbo gas (gas 2), nitrogen 30 psi.

2.3. Biosynthesis of fenoldopam-glucuronide and fenoldopam-sulfate

2.3.1. Mice and rat liver S9 fraction preparation.

liver S9 fraction or microsomes was prepared from C57/BL6 mice (Male, 8 weeks) or Sprague Dawley rat (Male, 8 weeks) according to the reported procedures and stored at −80 °C until used [14, 15].

2.3.2. Phase II metabolism reaction systems.

Biosynthesis of fenoldopam-glucuronide and fenoldopam-sulfate was conducted according to the protocol used in our lab previously [15, 16]. To synthesize fenoldopam-glucuronide, 10 μM fenoldopam was incubated with liver S9 fraction, MgCl₂ (5 mM), saccharolactone (4.4 mM), alamethicin (0.022 mg/ml), and UDPGA (3.5 mM) in a 50 mM potassium phosphate buffer (pH 7.4) for 2 hours (final S9 concentration = 2 mg protein/ml) at 37 °C. To synthesize fenoldopam-sulfate, 10 μM fenoldopam was typically incubated with PAPS and liver S9 fraction for overnight (final S9 concentration = 2 mg protein/ml) at 37 °C.

2.3.3. Metabolites purification.

Fenoldopam-glucuronide and fenoldopam-sulfate were purified using liquid-liquid extraction. Briefly, 10-fold of ethyl acetate was used to extract fenoldopam from the reaction solvent twice. Then the aqueous layers were dried under nitrogen flow and the residues were further purified to afford fenoldopam-glucuronide and fenoldopam-sulfate (>95 % UPLC-UV analysis).

2.3.4. Metabolites Identification.

The biosynthesized metabolites were identified using chemical and spectral methods. Briefly, the pure metabolites were incubated with glucuronidase or sulfatase, respectively, and analyzed in UPLC-UV. The retention time of the hydrolyzed product was compared to that of fenoldopam. In addition, the pure metabolites were subjected to LC-MS/MS analysis to afford fragments information and subjected to UPLC-UV analysis to afford UV spectra, which were used to compare with that of fenoldopam.

2.3.5. Quantification of fenoldopam-glucuronide and fenoldopam-sulfate.

The concentrations of fenoldopam-glucuronide and fenoldopam-sulfate in stock solutions were determined using a standard curve of fenoldopam and conversion factors of extinction coefficients according to the published protocol [17–20]. Briefly, the factors were determined by comparing the peak area increased in aglycone after hydrolysis by sulfatase or glucuronidase with the corresponding peak area decreased in metabolites. By plugging K values into the following equations using peak areas of metabolite, concentrations of metabolites CM were calculated by equation

$$\mathrm{CM}=\mathrm{C}/\mathrm{K}$$

Where C and CM are the concentrations of fenoldopam and fenoldopam metabolite, respectively, K is the conversion factor.

2.4. Method validation

2.4.1. Calibration curve and LLOD.

Calibration standards were prepared in 50 % methanol by diluting a stock solution of fenoldopam and fenoldopam-sulfate to final concentrations of 1,000.00, 500.00, 250.00, 125.00, 62.500, 31.25, 15.60, 7.80, 3.90, 1.95, 0.975, and 0.488 nM respectively. For fenoldopam-glucuronide, the concentrations of calibration curve were 10,000.00, 5,000.00, 2,500.00, 1,250.00, 625.00, 312.50, 156.00, 78.00, 39.00, 19.50, 9.75, and 4.88 nM respectively. To prepare standard curve in plasma, blank plasma (10 μL) was mixed with 10 μL of standard curve samples prepared in 50% methanol and 400 μL of internal standard, then sonicated for drug extraction. The 50 nM internal standard formononetin was dissolved in a mixture solvent (Methanol-Ethylacetate, 1:1, v/v). After centrifugation at 20,000 ×g for 15 min, the supernatant was transferred to a new tube and dried under a stream of N₂. The residue was reconstituted in 100 μL of 50% methanol and centrifuged at 20,000 g for 15 min for injection. The linearity of each calibration curve was determined by plotting the ratio of the peak areas of fenoldopam and its metabolites to the internal standard in rat plasma. A least-squares linear regression method (1/x² weight) was used to determine the slope, intercept and correlation coefficient of the linear regression equation. The lower limit of detection (LLOD) was defined based on a signal-to-noise ratio of 10:1.

2.4.2. Precision and accuracy.

The “intraday” and “interday” precision and accuracy of the method were determined with quality control (QC) samples at three different concentrations (three injections for each concentration) on the same day or on three different days. The low, middle and high effective concentration of QC samples for fenoldopam and fenoldopam-sulfate were 500.00, 62.50, 3.90 nM, and for fenoldopam-glucuronide was 5,000.00, 625.00, 78.00 nM, respectively.

2.4.3. Extraction recovery and matrix effect.

The extraction recoveries of fenoldopam and its two metabolites were determined by comparing the relative peak areas obtained from blank plasma extracts spiked with analytes, and those obtained from water spiked with the same amount of analytes. The matrix effect was studied by analyzing standard solution injected directly in mobile phase and comparing the response (peak areas) of drug and an internal standard (II) with the peak areas of standards spiked into the plasma extracts after extraction. These evaluations were performed according to the recommended validation procedures reported by FDA and EMA guidelines [21, 22].

2.4.4. Stability.

The stability of fenoldopam, fenoldopam-glucuronide, and fenoldopam-sulfate in rat plasma was determined by analyzing three replicates of QC samples at three different concentrations following storage at 25 °C for 6 h, at − 80 °C for 60 days, and after going through three freeze-thaw cycles (−80 °C and 25 °C). Stability was expressed as the ratio of the mean peak area (triplicate samples) of an analyte at different time points to the mean peak area of the same analyte at time zero multiplied by 100.

2.5. Pharmacokinetics study

2.5.1. Animals.

Female F344 rats were obtained from Harlan Laboratory (Indianapolis, IN) and kept in an environmentally controlled room (temperature: 25 ± 2°C, humidity: 50 ± 5%, 12 h dark-light cycle) for at least 1 week before the experiments. The rats were given ad libitum food and water. The animal protocols used in this study were approved by the University of Houston’s Institutional Animal Care and Use Committee.

2.5.2. Pharmacokinetics experimental design.

Fenoldopam was dissolved in saline and then given intravenously to rats at 2 mg/kg, the injection volume was 0.1 mL/100 gram. Blood samples (30–40 μl) were collected at 0, 0.25, 0.5, 1, 2, 3, 4, 6, and 24 hrs by snipping the tails under anesthesia with isoflurane. Plasma was separated by centrifugation (9,300 ×g, 8 min). The plasma samples were stored at −80 °C until analysis. The plasma samples were prepared at the same procedure as the blood samples for calibration according to part 2.4.1.

2.6. Data analysis and Statistical analysis

The data were analyzed by WinNonlin 3.3 software (Pharsight, Mountain View, CA) with non-compartmental models. The data were presented as mean ± SD, significance differences were assessed by using unpaired Student’s t-test. A p value of less than 0.05 or p<0.05 was considered as statistically significant.

3. Results and discussion

3.1. Chromatography and mass spectrometry

A specific, sensitive and reliable method to quantify fenoldopam and its major metabolites was established. To the best of our knowledge, this is the first analytical method that can quantify fenoldopam and its metabolites simultaneously. A typical MRM chromatogram of fenoldopam and its metabolites in rat plasma sample after IV administration of fenoldopam is shown in Fig.1. The retention time of fenoldopam-glucuronide, fenoldopam, fenoldopam-sulfate, and formononetin (IS) were 1.6, 1.9, 2.1 and 3.2 min, respectively.

Usually, the polarity of a sulfate is higher than that of the parent compound and retention time of sulfate is shorter than that of the parent compound in a chromatography with a reverse phase column such as C18 column. For fenoldopam, under the current elution using water/acetonitrile, the retention time of fenoldopam-sulfate is longer than fenoldopam (Fig. 1), probably because of a complex 3D structure change after sulfonation. More chemical studies are needed to explain this observation, which is out of the scope of this study.

For MS detection, we tried both negative and positive scan mode by infusing the analytes and tuning the compound- and instrument-dependent parameters (Table 1). The results showed that fenoldopam and glucuronide are more sensitive in the positive mode, while fenoldopam-sulfate was more sensitive in the negative mode. Therefore, we established a positive-negative switching method that allows us to quantify all analytes in a single injection. Positive-negative switching is a MS acquisition technique that allows a method with positive and negative ion switching to run without compromising data quality due to rapid polarity switching (e.g., < 5 milliseconds). With this novel technique, we will be able to run both positive and negative scan to quantify fenoldopam and its metabolites simultaneously while maintaining a high duty cycle, high selectivity, high sensitivity, and chromatographic peak shape integrity.

Table 1.

Compound-dependent parameters for fenoldopam and its two conjugates in MRM mode of UHPLC-MS/MS analysis.

Compound	Q1	Q3	DP	CE	CXP
Fenoldopam	306	107	146	33	13
Fenoldopam-glucuronide	482	306	120	30	13
Fenoldopam-sulfate (negative)	384	304	−64	−32	−13
IS (formononetin negative)	267	252	−100	−36	−15
IS (formononetin positive)	269	254	100	36	15

DP: Declustering potential, CE: Collision energy, CXP: Collision cell exit potential

3.2. Extraction recovery using different solvents

Due to polarity difference, it is challenging to extract fenoldopam and its glucuronide and sulfate simultaneously as reported previously [9–12]. In this study, we tried different solvents to improve the extraction recovery. The results showed that when acetonitrile or methanol was used alone, only metabolites can achieve the satisfied extraction recovery (i.e., >80%), while when acetate ethyl was used alone, only fenoldopam can be efficiently extracted. Then, we developed a mixture of methanol-ethyl acetate (1:1, v:v) to achieve good recovery for both parent and metabolites (Table 3).

Table 3.

Extraction recovery (%) for fenoldopam and its two conjugates with different mixture solvent (n=3).

Analyte	QC concentration (nM)	MeOH (%)	ACN (%)	Ethyl Acetate (%)	Methanol-Ethylacetate (1:1, v/v)(%)
Fenoldopam	3.90	0 ±0	0 ± 0	101.5 ± 2.1*	83.1 ± 7.5*
	62.50	0 ± 0	0 ± 0	102.2 ± 3.4*	82.0 ± 10.5*
	500.00	5.30 ± 0.5	3.1 ± 0.4	97.0 ± 4.1*	81.3 ± 4.1
Fenoldopam - Glucuronide	78.00	111.5 ±8.2	116.4 ± 12.1	3.2 ± 0.8*	103.2 ± 3.1*
	625.00	104.3 ± 6.7	86.7 ± 3.0	7.3 ± 2.5*	96.7 ± 6.7*
	5,000.00	103.3 ± 5.5	115.8 ± 3.1	7.9 ± 3.7*	93.6 ± 6.4*
Fenoldopam -Sulfate	3.90	108.2 ± 6.2	90.9 ± 8.7	30.2 ± 5.7	113.9 ± 13.2
	62.50	91.4 ± 8.4	100.1 ± 6.3	20.5 ± 9.2	109.9 ± 8.6
	500.00	90.3 ± 10.4	89.5 ± 9.2	24.2 ± 9.5	111.9 ± 7.6

ACN: acetonitrile, MeOH: methanol

^*means compared with those of ACN

^#means compared with those of methanol, p<0.05.

3.3. Identification of the metabolites

The two metabolites were generated from glucuronidation and sulfonation reactions, suggesting that the metabolites are fenoldopam-glucuronide and -sulfate, respectively. Additionally, when the two metabolites were incubated with glucuronidase or sulfatase, the peaks in UPLC-UV analysis was disappeared and an additional peak was observed at the same retention time as that of fenoldopam, confirming that these two metabolites are fenoldopam-glucuronide and -sulfate, respectively. In UPLC-UV analysis, the UV spectra of the two metabolites are similar to that of fenoldopam (Fig. 2) In MS/MS analysis, the two metabolites release fragments at m/z 176 or 80, which are typical fragments of glucuronide and sulfate, respectively. Based on these findings, these two metabolites were identified as fenoldopam-glucuronide and fenoldopam-sulfate. The position of the glucuronide and sulfate was not determined in this study. More metabolites (mg range) are required to run NMR for position determination.

Figure 2.

The UV spectra and MS spectra of fenoldopam-sulfate and fenoldopam-glucuronide. The UV profile of fenoldopam (blue line) and fenoldopam-sulfate and fenoldopam-glucuronide (red line and purple line) determined by a DAD detector from 200 nm to 400nm (Fig 3A). The MS spectra and fragmentation pathways of fenoldopam-sulfate and fenoldopam-glucuronide. in positive (Fig 2B) and negative modes (Fig 2C).

3.4. Method validation

3.4.1. Linearity and lower limit of detection (LLOD)

The standard curve of fenoldopam-glucuronide was linear from 9.75 to 10,000.00 nM (R > 0.99) and from 0.98 to 1,000.00 nM for fenoldopam and fenoldopam-sulfate in rat plasma (Table 2). The accuracy and reproducibility of these measurements are shown in Table 2 and were found to be in the acceptable range (85–115%) according to FDA guidance. The intraday and interday variance of QC samples were less than 8.4 % and the accuracy were between 82.5–116.0 %. LLOQ of fenoldopam, fenoldopam-glucuronide and fenoldopam-sulfate is 0.98, 9.75 and 0.98 nM, respectively. A representative chromatogram at LLOD is shown in Fig. 3.

Table 2.

Standard Curve, linear range, and LLOQ for Ral and its two conjugates in MRM mode of UHPLC-MS/MS analysis.

Analyte	Concentration(nM)	Curve	R	LLOQ (nM)
Fenoldopam	1,000–0.98	Y=0.00429X −0.00815	0.9656	0.98
Fenoldopam-glucuronide	10,000–9.75	Y=0.000693X − 0.000927	0.9765	9.75
Fenoldopam-sulfate	1,000–0.98	Y=0.00102X + 0.000605	0.9917	0.98

Figure 3.

Representative MRM chromatograms of (A) blank plasma spiked with the fenoldopam and its two metabolites at LLOQs and (B) blank plasma.

3.4.2. Accuracy and precision

Accuracy and precision were determined by quantifying six replicates of QC samples at three QC concentration levels and the LLOQ in pooled blank rat plasma according to the FDA guidance. The precision and accuracy are shown in Table 4. These results demonstrated that the precision and accuracy values were in the acceptance range.

Table 4.

Precision, accuracy, and matrix effect of fenoldopam and its two conjugates in MRM mode of UHPLC-MS/MS analysis.

Analyte	Concentration of QC samples (nM)	Matrix effect	Precision
			Intraday		Interday
		Average ± SD (%)	Accuracy (%)	Precision (RSD, %)	Accuracy (%)	Precision (RSD, %)
Fenoldopam	0.98	95.0 ± 8.9	93.3	7.7	97.6	5.3
	3.90	102.0 ± 4.6	108.6	3.8	107.9	4.3
	62.50	100.7 ± 9.8	111.5	3.7	90.9	8.4
	500.00	99.4 ± 3.5	116.0	8.4	94.5	4.1
Fenoldopam-glucuronide	9.75	83.7 ± 5.9	95.4	7.9	84.1	5.5
	78.00	108.3 ± 7.0	103.9	1.7	82.5	4.6
	625.00	90.2± 5.9	106.9	4.7	85.5	3.8
	5,000.00	84.8 ± 9.2	109.0	7.0	94.4	3.8
Fenoldopam-sulfate	0.98	84.2 ± 7.8	85.3	6.1	88.3	4.4
	3.90	88.7 ± 11.0	99.5	2.5	83.5	5.4
	62.50	88.4 ± 5.9	109.1	2.8	87.0	8.0
	500.00	89.6 ± 6.3	115.7	1.6	89.9	1.7

3.4.3. Matrix effect and stability

The matrix effect and stability were determined using fenoldopam and its metabolites in the QC samples at three concentration levels. The relative peak areas of these three analytes after spiking into blank rat plasma were comparable to those spiking to water, suggesting that the matrix effect of fenoldopam, fenoldopam-glucuronide and fenoldopam-sulfate were in acceptable ranges (Table 4).

The stability of fenoldopam and its two metabolites in rat plasma was evaluated by analyzing three replicates of QC following storage at 25 °C for 6 h, at − 80 °C for 60 days, and after going through three freeze-thaw cycles (from −80 °C to 25 °C). The recovery of fenoldopam and its two metabolites was found to be 86.1–113.9% in all the conditions tested (Table 5), indicating that the stability of these three analytes were in acceptable range under the above storage condition.

Table 5.

Stability of fenoldopam and its metabolites in rat plasma under different storage conditions (n=3)

Analytes	Spiked Concentration (nM)	25°C for 6 hours	Three-freeze-thaw cycles	Frozen for 2 months
		Accuracy (RE, %)	Accuracy (RE, %)	Accuracy (RE, %)
Fenoldopam	3.90	13.0	12.3	12.5
	62.50	13.5	11.3	12.4
	500.00	11.5	12.6	9.8
Fenoldopam-glucuronide	78.00	8.4	10.5	7.5
	625.0	11.4	−10.7	7.7
	5,000.00	10.9	11.0	7.1
Fenoldopam -Sulfate	3.90	4.2	5.2	−5.3
	62.50	−0.9	11.9	−0.9
	500.00	10.3	7.1	11.4

3.5. Application in a pharmacokinetics study in rats.

The dose of fenoldopam was 2.0 mg/kg, which is the human equivalent dose. The mean plasma concentration-time curves of fenoldopam and fenoldopam-glucuronide and fenoldopam-sulfate after IV administration were presented in Fig. 4 and the PK parameters were listed in Table 6. The results showed that the concentration of fenoldopam in the plasma decreased rapidly after injection with a terminal half-life (t_1/2) of 0.63 ± 0.24 h, which is consistent with that in humans. The AUC0-t and Cmax of fenoldopam were 13.36 ± 5.16 nM and 7.72± 2.03 nmol h/L, respectively. The PK parameters of fenoldopam-sulfate were similar with those of the parent. The t_1/2, AUC0-t and Cmax of fenoldopam-sulfate was 0.48 ± 0.19 h, 65.18 ± 13.13 nM, and 68.58 ± 19.57 nmol h/L, respectively. In contrast to the fenoldopam and fenoldopam-sulfate, fenoldopam-glucuronide concentration in the plasma is significantly higher. The t_1/2 of fenoldopam-glucuronide was 35-fold longer than parents, and the AUC0-t and Cmax was 3900- and 1000-fold higher than fenoldopam. The observation suggested that fenoldopam was rapidly metabolized into glucuronide in vivo to cause a short half-life. Manipulating glucuronidation could be a possible approach to prolong the duration of the treatment.

Figure 4.

Time vs. blood concentrations fenoldopam, fenoldopam-glucuronide, fenoldopam-sulfate after IV administration fenoldopam (2 mg/kg, n=5) to rats.

Table 6.

Pharmacokinetic parameters of fenoldopam and its two conjugates intravenous administration of fenoldopam (2 mg/kg) to rat (n=5)

Parameters	Fenoldopam	Fenoldopam-glucuronide	Fenoldopam-sulfate
Tmax(h)	0.25 ± 0.00	0.25 ± 0.00	0.35 ± 0.14
Cmax(nmol/L)	13.36 ± 5.16	14633.30 ± 4202.78	65.18 ± 13.13
AUC0∼t(nmol h/L)	7.72± 2.03	30171.20 ± 9651.18	68.58 ± 19.57
AUC0∼∞(nmol h/L)	7.72± 2.03	30453.80 ± 10100.60	68.58 ± 19.57
MRT(h)	0.50 ± 0.03	5.10 ± 1.58	0.57 ± 0.15
T1/2(h)	0.63 ± 0.24	21.90 ± 11.67	0.48 ± 0.19
CL(L/h/kg)	542.49 ± 71.03	0.17 ± 0.09	91.99 ± 27.66
Vz(L/kg)	495.02± 224.03	4.45 ± 1.27	60.55± 18.95

4. Conclusion

To the best of our knowledge, this is the first validated analytical method to simultaneously quantify fenoldopam and its major metabolites in the plasma. This method is robust and sensitive. The one-step protein precipitation using a mixture solvent (methanol and ethyl acetate) can efficiently extract fenoldopam and its metabolites. This method is also valuable for human clinical study because it allows for even higher sensitivity than reported here since a large blood volume is usually available and thereby may be used to concentrate the analyte before analysis.

1. An UPLC-MS/MS method to quantify fenoldopam was developed and validated.

2. The method can quantify fenoldopam and its conjugates simultaneously.

3. The sensitive and robust method was used in a PK study in rats.

Abbreviations:

UHPLC

ultra high performance liquid chromatography

IS

internal standard

AUC

area under the curve

QC

quality control

LLOQ

lower limit of quantification

PK

pharmacokinetic

FDA

federal drug administration

IV

intravenous

SD

Sprague Dawley rat

LLOD

lower limit of detection

RE

Relative error

RSD

relative standard deviation