Analysis of Pz-1, a Promising Therapeutic for Organophosphorus Poisoning from Rodent Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Organophosphorus (OP), pesticides (e.g., parathion) and nerve agents (e.g., soman) can produce acute and long-term neurological problems. Exposure to OP chemicals is responsible for an estimated 200,000 deaths annually. Pz-1 (N-(5-(tert butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide) is a muscle specific kinase (MuSK) inhibitor which has shown potential as a treatment for OP chemical exposure. While development of this treatment requires the availability of a validated analytical method, no method currently exists for analysis of Pz-1 from biological samples. In this study, an analytical method was developed for Pz-1 from rat (and mouse) plasma. Plasma was prepared by precipitating plasma proteins, isolating the supernatant, evaporating to dryness and reconstituting in 1:1 MeOH:water. Prepared samples were analyzed by reversed-phase liquid chromatography tandem mass-spectrometry (LC-MS/MS). The method produced excellent sensitivity, with a limit of detection of 1 nM (455 ng/L). The calibration range was 3 – 100 nM and the calibration curve produced excellent linear behavior (R² ≥ 0.99 and PRA ≥ 91%). The method also showed good accuracy and precision. The validated method was used to detect Pz-1 in mouse plasma following intraperitoneal (IP) treatment with 5 mg/kg Pz-1. In summary, this method shows promise as a simple and sensitive method to analyze Pz-1 in rat plasma to facilitate its continued development as a treatment for OP toxicity.

1. Introduction

Organophosphates (OPs) have been utilized as pesticides (e.g., malathion) and chemical warfare agents (e.g., VX). Humans are most commonly exposed to OP-based herbicides, killing almost 200,000 people annually, primarily in rural parts of underdeveloped countries [1–6]. Additionally, OP-based chemical warfare agents have been used in armed conflicts, including the Iran-Iraq war, the Syrian civil war, and terrorist actions (e.g., the Tokyo subway sarin assault) [1, 3, 7]. OPs primarily target acetylcholinesterase (AChE), a serine hydrolase present in brain synapses, neuromuscular junctions (NMJs), and red blood cells [1, 4–9]. OP compounds bind covalently to AChE at a catalytic serine residue and inhibit the active site of the enzyme. Inhibiting AChE can create a cholinergic crisis, where acetylcholine accumulates at synapses and NMJs [1, 5, 7–9]. When excessive quantities of acetylcholine accumulate, extreme toxic outcomes ensue [2, 7, 9, 10]. Both nicotinic and muscarinic toxicity can occur simultaneously due to the excessive accumulation of acetylcholine [10]. Nerve impulses fire continuously, leading to increased parasympathetic activities and involuntary muscle spasms. Symptoms of OP intoxication include cramping, increased salivation, lacrimation, paralysis, muscular fasciculation, weakening in the muscles, diarrhea, and blurred eyesight [10]. This can lead to seizures or respiratory failure [1, 5]. Also, OP toxicity can damage the central nervous system and disrupt hormone production in humans and animals [10, 11].

OP poisoning is currently treated with a combination of anticholinergics (e.g., atropine) and oximes (e.g., 2-PAM) [1, 2, 6, 7, 9, 12]. Atropine inhibits cholinergic receptors, whereas oximes, or their deprotonated counterpart, replace the OP ligand on the serine at the enzyme’s active site, thereby reactivating the OP-inhibited AChE [1, 3]. Although various oximes have been shown to be effective at treating OP poisoning, none are 100% effective against all OPs [1, 12, 13]. This is typically because dealkylation of the OP-AChE ligand occurs when a second leaving group is lost from the OP ligand, resulting in an oxyanion on the phosphoryl group [1, 13]. This process is known as aging of AChE [1, 2, 13]. The aged form of AChE is resistant to reactivation by any known oxime because the electrostatic connection between the oxyanion and positively charged catalytic histidine stabilizes the structure of aged AChE, rendering it resistant to reactivation [1, 13]. Victims exposed to fast-aging OPs, such as soman (with a half-life (t_1/2) of several minutes), have a low chance of survival [1, 13]. Despite the discovery of aging in the 1950s, no clinical treatments have been adequately established to restore the function of aged AChE [1, 2, 13]. Most current research on OP poisoning is still focused on therapies for non-aged AChE, including several oximes which are used to reactivate AChE but are not applicable against all OP compounds and have the additional shortcoming of insufficient blood brain barrier permeability [1, 2, 13]. Therefore, there is a critical need to develop strategies to accelerate the turnover of aged AChE.

One promising molecule shown to help increase the AChE turnover rate is Pz-1(N-(5-(tert butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)phenyl)acetamide). Pz-1 is a cell-permeable benzimidazole. It is a non-cytotoxic and highly potent type II kinase inhibitor that affects tyrosine kinase activity to impede the growth of cancer cells [14–18]. Pz-1 has been demonstrated to inhibit VEGFR2 (i.e., vascular endothelial growth factor receptor 2) which subsequently inhibits the growth of blood vessels, cutting off nutrient supply to tumors [14–16]. Pz-1 (at 1.0 mg/kg/day) was shown to prevent tumor growth generated by RET-mutant fibroblasts and inhibits phosphorylation of both RET and VEGFR2 in tumor tissue [15, 16]. Pz-1 exhibits no observable toxicity up to 100.0 mg/kg, indicating a broad therapeutic window [15, 16]. While Pz-1 has been predominantly developed as a cancer treatment, it is also a known inhibitor of muscle-specific protein kinase (MuSK), which has been shown to induce turnover of aged AChE [2, 19]. The inhibition or loss of MuSK destabilizes the AChE complex, resulting in the enzyme’s destruction. This releases the aged enzyme, allowing fresh AChE to repopulate the synapse, restoring optimum neurotransmission, and minimizing the consequences of rapid-aging OP exposures [2, 19].

Despite the potential advantages of Pz-1 as a MuSK inhibitor and cancer treatment, there is currently no analysis method for quantifying Pz-1 in any biological matrix. For the development of Pz-1 as a viable therapeutic, a validated analytical method is required. As a result, the goal of this study was to create and validate an effective liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique for analyzing Pz-1 in rodent plasma, allowing for continued development of Pz-1 as a treatment against several rapid-aging OP compounds, such as soman (2-min) and di-isopropyl fluorophosphate (DFP, 5-h) [1, 2, 13, 20, 21].

2. Materials and Methods

2.1. Reagents and standards

Ammonium formate, methanol (MeOH), and acetonitrile (ACN) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Dimethyl sulfoxide (DMSO) and Pz-1 were acquired from Sigma-Aldrich (St. Louis, MO, USA). Using a Lab Pro polishing unit from Labconco Corporation (Kansas City, KS, USA), reverse-osmosis water (DI water) was purified to a resistivity of 18.2 MΩ-cm. Unless specified otherwise, all of the reagents employed in this experiment were of the high-performance liquid chromatography (HPLC) grade. Tween 20 was obtained from Millipore Sigma (St. Louis, MO, USA).

Pz-1 stock solutions (0.55 mg mL⁻¹) were prepared in a 4-mL vial in 100% DMSO and maintained at −20 °C. The stock solution was diluted to 100 μg mL⁻¹ in 80:20 DMSO:water. Pz-1 standards for calibration and quality control (QC) were diluted with 50:50 MeOH:water using successive dilutions to achieve the desired concentration. When needed, this solution was spiked into rat plasma to produce Pz-1 standards. Prior to sample preparation, Pz-1 spiked plasma was well mixed for 60 s at 3,000 rpm (252 × g). Diluent, Tween 20% (v/v), was prepared in 80% sterile filtered water (RNase-free, deionized, not-DEPC treated). Diluent (600 μL Tween 20 and 2.4 mL water) was prepared and sterile filtered through a syringe filter. Diluent alone was used as vehicle control (VC) for the animal studies.

2.2. Biological samples

For method development, rat plasma was acquired from BioIVT Elevating Science (Westbury, NY, USA) and kept at −80 °C until needed. To evaluate the effectiveness of the analytical method, mice were treated with Pz-1 and plasma was later obtained and analyzed. Table 1 lists the important characteristics of the mice used in this study and other important experimental parameters. Mice were administered 5 mg/kg Pz-1 by body weight or the corresponding volume of vehicle control (20% Tween 20 in water). Intraperitoneal (IP) injections were performed using a 27-gauge needle. Blood was collected at two time points: 30-min and 60-min post-treatment. Microcentrifuge tubes (2-mL) were prepared for cheek blood collection by filling them with 500 μL heparin solution (10–20% w/w) and shaking them for 2 min by hand. Just before blood collection for each mouse, the tube was shaken again, and the heparin solution was removed. Heparin solution (50 μL) was added to the tube to mix with the blood. For the 30 min blood draw, a lancet (5 mm) was used for a cheek bleed. Blood was collected directly into the heparin-coated microfuge tube. The tube was capped, flicked gently to mix, and immediately put on ice. Saline (300 μL s.c.) was administered after the cheek bleed on the left flank. After 30 min, blood samples were centrifuged at 1000 × g for 6 min at room temperature. Plasma was transferred to new 2-mL microfuge tubes (no additional heparin was added). The remaining blood was centrifuged again at 2000 × g for 8 min to separate and collect any remaining plasma. Plasma was kept on ice. Blood was also collected at approximately 60 min (62–71 min) post-IP-injection by cardiac puncture. For this collection, mice were euthanized by CO₂ inhalation and cervical dislocation. The heart was exposed and blood was drawn into a 1-mL tuberculin syringe that had been precoated with heparin solution by drawing up 1-mL and expelling it. The heparin in the headspace was left to mix with the blood. Blood was collected from the left ventricle using a 27 g needle. After collection, the needle was removed and blood was gently expelled from the syringe into a 2-mL microfuge tube prepared as described above for the cheek bleed, with the exception that the heparin solution (50 μL) was not added to these tubes due to the heparin already in the needle headspace. Cardiac (60-min time point) whole blood was centrifuged at 2000 × g for 8 min at room temperature. Plasma was transferred to a new 2-mL microfuge tube (not heparin-coated and with no additional heparin) and kept on ice. The plasma was clear to slightly yellowish, indicating minimal hemolysis. Samples were stored at −80°C until analyzed. In compliance with NIH rules, the Institutional Animal Care and Use Committees of the South Dakota State University authorized all experimental procedures.[22]

Table 1.

Characteristics of animals treated with Pz-1 vs. VC with dosing and blood collection information.

Mouse	Sex	Weight(g)	IP Volume(μL)	Pz-1/VC	Blood Volume (μL)
					30 min	60 min
1	M	31.0	155	VC	100	~275
2	F	23.3	117	VC	150	~225
3	M	27.9	140	Pz-1	150	~300
4	F	22.7	114	Pz-1	200	~250

2.3. Sample preparation for LC-MS/MS analysis

Different concentrations of Pz-1 (50:50 H₂O:MeOH) were spiked into rat plasma to produce calibration or QC standards. For prepared standards, an aliquot (200 μL) of spiked or unspiked rat plasma was added to a 2-mL microcentrifuge vial. ACN at a 1:8 ratio (1.6 mL) was added to precipitate plasma protein. The mixture was vortexed for 20 s at 3,000 rpm (252 × g) and cold centrifuged for 10 min at −8 °C and 14,800 rpm (15339 × g). Subsequently, the supernatant from the centrifuge tube was transferred into a 4-mL glass vial and dried at room temperature for approximately 30 min under a stream of inert nitrogen gas. After the sample was completely dried, it was reconstituted using 200 μL of 50:50 H₂O:MeOH. It was then vortexed for approximately 10 s at 3000 rpm (252 × g), and a 0.22 μm nylon syringe filter was used for filtering the solution. The filtrate (~200 μL) was placed into a glass insert inside a 2-mL HPLC vial for LC-MS/MS analysis. Because the mouse plasma sample volumes were very low (~150 μL), an accurate volume of plasma was aliquoted into a clean vial and the remainder of the sample preparation steps were performed as described, including reconstitution in 200 μL of 50:50 MeOH:H₂O. Accurate concentrations of Pz-1 for the animal study were calculated by accounting for the initial volume differences.

2.4. LC-MS/MS analysis

A Shimadzu HPLC system (20 ADXR, Shimadzu Corp., Kyoto, Japan) was used for LC-MS/MS analysis of Pz-1. An Ascentis LC-RP column (10 cm length × 3 mm internal diameter, 2.7 μm particle size, and 90 Å pore size) was utilized for liquid chromatographic separation of Pz-1. Prepared samples were kept in a 15 °C cooled autosampler. Pz-1 (3-μL injection volume) was separated using a flow rate of 0.4 mL/min with a gradient of 65% B increased linearly to 100% over 1 min, held constant for 2.5 min, decreased to 65% B over 0.5 min, and held constant for 2 min to equilibrate between injections. Ammonium formate (5 mM) in 99:1 water:ACN (mobile phase A) and ammonium formate (5 mM) in 95:5 ACN:water (mobile phase B) were used as mobile phases. Carryover was initially a problem for Pz-1. Therefore, to reduce carryover, after each analysis where significant concentrations of Pz-1 were expected (>25 nM), the LC system (including the column) was purged by performing two LC-MS/MS analyses: 1) analysis of a 50:50 MeOH:water solution and 2) analysis of 100% water. These chromatograms were evaluated for residual Pz-1 to ensure carryover was not present.

Detection of Pz-1 was accomplished using a tandem MS in positive polarity electrospray ionization (ESI) mode (AB Sciex QTRAP 5500 MS, Framingham, MA, USA). The curtain nitrogen gas pressure was 10 psi, with a source temperature of 500 °C, and an ion spray voltage of 4,500 V. The gas pressures for the nebulizer (GS1) and heater (GS2) were 10 and 0 psi, respectively. The collision energy was set at “medium” (med) for the method. Analyst software (Applied Biosystems, Version 1.6.3) was used to evaluate the mass spectra and LC-MS/MS chromatograms.

2.5. Calibration, quantification, and limit of detection

The FDA’s bioanalytical technique validation criteria were followed in order to validate the current method [23]. In order to assess the calibration behavior of Pz-1, triplicate calibration standards (1.5, 3, 6, 12, 24, 50 and 100 nM) were prepared in rat plasma. These samples were analyzed and the Pz-1 peak areas were plotted as a function of plasma Pz-1 concentration. To determine the best model for Pz-1 calibration, both weighted (1/x and 1/x²) and unweighted linear (y = mx + b) regression models were evaluated. The proposed method used a geometric series of concentrations for calibration curve preparation; hence, percent residual accuracy (PRA) was taken into consideration and prioritized over the commonly used coefficient of determination (R²) to measure the goodness-of-fit (GoF) of the calibration models [24].

The calibration range, including the upper and lower limits of quantification (ULOQ and LLOQ, respectively), was determined based on the series of calibrators with accuracies of 100±20% of the nominal calibrator concentration and precision of ≤15% relative standard deviation (RSD). To determine the limit of detection (LOD), multiple Pz-1 concentrations below the limit of quantification (LLOQ) were analyzed. The lowest concentration of Pz-1 that consistently resulted in a signal-to-noise (S/N) ratio of three was selected as the LOD. Noise was quantified as peak-to-peak noise of the blank over the elution time of Pz-1.

2.6. Accuracy and precision

For three separate days (within seven calendar days), calibrators were prepared in rat plasma each day and examined to determine the intra- and interassay accuracy and precision of the method. While the interassay accuracy and precision were computed by comparing the QCs over three different days, the intraassay accuracy and precision were ascertained from the QCs on each day’s analysis. Precision and accuracy goals were set at ≤ 15% RSD and 100±20%, respectively.

2.7. Matrix effect and recovery

Two calibration curves were created by adding Pz-1 to both plasma and aqueous solution. This allowed for comparison of calibration slopes of the plasma and aqueous matrix to determine the matrix effect. The effects of plasma matrix suppression or enhancement are indicated by slope ratios (i.e., m_plasma/m_aqueous) of less than or greater than one, respectively. The recovery was assessed by comparing the peak area of the QCs (low, medium and high) prepared as described and Pz-1 spiked in prepared blank plasma. The average peak area of Pz-1 prepared in plasma was divided by the average peak area of Pz-1 spiked in prepared blank plasma, and the result was multiplied by 100% to estimate recovery.

2.8. Stability

By examining triplicates of plasma spiked at low (5 nM) and high (30 nM) QC concentrations that were kept under various temperatures, the short and long-term stability of Pz-1 was assessed. When the signal from a stability standard was within ±20% of the original (time 0) signal, Pz-1 was deemed stable. Benchtop and autosampler stabilities were evaluated for 24 h as part of the short-term stability study. Pz-1 spiked non-denatured plasma for both QCs was placed on the workbench for 0, 1, 2, 5, 10, and 24 h at room temperature prior to analysis in order to measure bench-top stability. Prepared QCs were stored in the LC autosampler at 15 °C to assess the autosampler stability of Pz-1. They were analyzed immediately and at 0, 1, 2, 5, 10 and 24 h. For the long-term stability study, the Pz-1-spiked plasma samples were kept at room temperature, 4°C, −20°C and −80 °C for 0, 1, 2, 5, 15 and 30 days. Six sets of QC samples were generated in triplicate and one was analyzed immediately on “Day 0” of the experiment. All others were placed in their designated storage environment until analyzed. Frozen samples were thawed unassisted at room temperature. The stability of Pz-1 was quantified using the ratio of average peak areas of the QC stored at different temperatures to the QC analyzed immediately and calculated as a percentage.

3. Results and Discussion

3.1. LC-MS/MS analysis of Pz-1

To analyze Pz-1 via MS/MS detection, its fragmentation pattern was analyzed following ionization. The molecular ion of Pz-1 ([M+H⁺], m/z 454.9) was detected by the infusion of Pz-1 (in 80:20 DMSO: H₂O) into the ESI. Figure 1 displays the Q1 mass spectrum of Pz-1 generated by positive ESI (A) and product ion scan (B). As expected, the Pz-1 produced a strong molecular ion signal, well above other smaller MS fragments. Therefore, a product ion scan of the Pz-1 molecular ion was performed and two major fragments, 288.2 and 315.0 m/z, were identified (Figure 1B). The most abundant fragment, 315 m/z, occurred due to the cleavage of the amide C-N bond, losing the amine fragment and the 288.2 m/z fragment was formed due to the cleavage of the C-C bond opposite the C-N bond of the amide, losing the amide fragment. The 315.0 m/z fragment was over 10 times more abundant than the 288.2 m/z fragment. Therefore, the identification transition was selected as 454.9→288.2 m/z and the quantification transition was 454.9→315.0 m/z. The transitions were optimized for multiple reaction monitoring (MRM) analysis, and important MRM parameters are listed in Table 2.

Figure 1.

ESI(+) mass spectra obtained via (A) Q1 scan of Pz-1 with a clear major peak at 454.9 m/z, corresponding to the molecular ion [M+H]⁺ of Pz-1, and (B) product ion scan of m/z 454.9, Pz-1 molecular ion. Inset: structure of Pz-1 with likely fragmentation indicated for the most abundant fragments.

Table 2.

MRM transitions, and optimized declustering potentials (DPs), collision energies (CEs), and collision cell exit potentials (CXPs) for Pz-1 analysis by LC-MS/MS.

Use	Q1 (m/z)	Q3 (m/z)	Time (ms)	DP (V)	CE	(V)	CXP (V)
Quantification	454.9	315.0	100	245.98	49	06	23.09
Identification	454.9	288.2	100	145.99	54	02	30.00

The MS/MS parameters were integrated into an LC-MS/MS method which featured simple sample preparation, excellent selectivity and good sensitivity. This LC-MS/MS method is the first method published for Pz-1 from any biological matrix. Using this method, plasma samples are quickly and easily prepared and analyzed. The sample preparation features only 5 steps: 1) denaturing the plasma protein (1:8 ACN ratio), 2) centrifuging, 3) drying the supernatant (under inert nitrogen), 4) reconstitution (50:50 H₂O: MeOH), and 5) filtering with 0.2 μm syringe filter. On average, samples can be prepared in under 60 min. Moreover, the analysis of these prepared samples is rapid, with a chromatographic analysis time of only 3 min. Therefore, the entire method can be performed in less than 1 h. This rapid method allows an estimated 480 samples to be analyzed in parallel during a 24-h period, although carryover from samples containing large concentrations of Pz-1 may lengthen the analysis of multiple samples.

Chromatograms of the quantification and identification transitions from the analysis of plasma spiked with Pz-1 and non-spiked plasma are displayed in Figure 2. The Pz-1 peak eluted at 1.48 min is sharp with only slight tailing (T_f = 1.25, A_s = 1.5). Pz-1 is the only notable peak in the chromatogram. Additionally, the signal of the non-spiked plasma for Pz-1 over the elution time of Pz-1 can be considered baseline noise (Figure 2 inset). This result demonstrates the excellent selectivity of the method.

Figure 2.

LC-MS/MS chromatogram of Pz-1-spiked plasma (100 nM) and non-spiked plasma. Transitions 454.9→315 and 454.9→288.2 m/z are used for the represent quantification and identification transition, respectively. Inset: Chromatogram from non-spiked plasma at 3×10⁴ times magnification.

3.2. Dynamic range and limit-of-detection

The limit-of-detection (LOD) of the method was 1 nM (455 ng/L) in rat plasma. This LOD is excellent and allows for an extremely sensitive detection of Pz-1 for drug development studies. The low LOD also gives the method the ability to analyze smaller volumes of plasma (e.g., studies using mice) without losing the ability to detect Pz-1.

The dynamic range of the method was assessed using a concentration range of 1.5 nM – 100 nM. The lowest concentration (1.5 nM) did not meet the accuracy and precision criteria, so the final linear range was 3–100 nM (Note that 100 nM was the highest concentration calibrator evaluated). A weighted linear least squares (1/x²) regression model yielded the best fit for the calibration data based on the evaluation of calibration curve residuals. Three calibration curves were generated over the course of three days in order to assess the calibration’s consistency. Important parameters for the calibration curves are reported in Table 3. All calibration curves displayed satisfactory goodness-of-fit PRAs > 91% and R² ≥ 0.99, indicating an excellent fit of all the data over the calibration range. Generally consistent slopes and intercepts were obtained, with <15% relative standard deviation between both the slopes and intercepts over the 3 days. Although the calibration curve is generally consistent over 3 days, it is still recommended that calibration curves be prepared fresh during each day of analysis since there is currently no internal standard available for the method.

Table 3.

Calibration curve equations prepared on three consecutive days with related goodness-of-fit parameters.

Day	Calibration equation	PRA (%)	R2
1	y = 14012× - 6192	91.0	0.9939
2	y = 11655× - 5677	96.9	0.9975
3	y = 15663× - 4593	96.3	0.9992

3.3. Accuracy and Precision

To evaluate the precision and accuracy of the method, a quintuplicate analysis of three (low, medium, high) QCs (5, 15, 30 nM) was conducted over the course of three different days. As shown in Table 4, the aggregate inter- and intraassay accuracies and precisions are within 100±15% and <16% RSD respectively. Note that only one intraassay precision value was above 15%. The accuracy of the method was especially good, with accuracies ranging from 100±2% to 100±12%. Considering no internal standard was utilized for this method, the accuracy compares favorably to other bioanalytical methods [25–27].

Table 4.

The intra- and interassay accuracies and precisions for analysis of Pz-1 from spiked rat plasma.

	Intraassay						Interassay
Conc (nM)	Accuracy (100±x%)a			Precision (%)b			Accuracy (100±%)a	Precision (%)b
	Day 1	Day 2	Day 3	Day 1	Day 2	Day 3
3 (LLOQ)	11	11	3.1	2.8	6.0	10.7	8.4	<6.6
5 (Low)	9	4	2	10.9	7.11	14.7	5	<10.8
15 (Med)	9	8	10	15.4	7.06	5.62	9	<9.2
30 (High)	4	12	9.6	11.9	12.1	11.7	2.1	<11.8

^aQC method validation (N = 5).

^bMean of three different days of QC method validation (N = 15).

3.4. Matrix effect, recovery, and storage stability

By comparing the slopes of the plasma and aqueous calibration curves (i.e., m_plasma/m_aqueous), the matrix effect was quantified. The slope ratio was 0.62, meaning that there is a significant matrix effect from plasma. For low, medium, and high QCs, the percent recoveries for Pz-1 in rat plasma were 71.7, 97.7, and 80.9%, correspondingly. The overall recovery was generally good for such a simple sample preparation scheme. Entrapment of Pz-1 in the plasma precipitate is the likely cause of recoveries lower than 90% and the inconsistency in the recovery values. While some recoveries were < 90%, this had little bearing on the precision or accuracy of the Pz-1 analysis (Table 4).

Pz-1 showed remarkable stability. Pz-1 was stable in spiked non-denatured plasma on the bench-top. The signal recoveries for the high and low QCs were above 80% of the “time 0” signal at all-time points. Also, prepared samples were stable on the autosampler over the course of the study. Sample stability was also evaluated at multiple storage conditions (4, −20 and −80°C) for 0, 1, 2, 5, 15, and 30 days. Pz-1 samples stored at different storage conditions remained stable for 30 days. Based on the excellent stability of Pz-1 in plasma under all conditions, it is only important to consider the stability of the plasma itself (including clotting potential). Therefore, it is recommended to store plasma samples at 4°C if medium-term storage is necessary and at −20 or −80°C if long-term storage is necessary. For short-term storage, any storage condition is applicable.

3.5. Analysis of Pz-1 from plasma of treated mice

Plasma from Pz-1 treated mice was analyzed using the validated LC-MS/MS technique. Chromatograms of mouse plasma obtained from mice treated with Pz-1 (5 mg/kg IP) and vehicle are displayed in Figure 3. In the treated plasma samples, the Pz-1 peak was detected at 1.45 min, but the non-treated mouse plasma did not exhibit any signal above baseline noise at the Pz-1 retention time (Figure 3, inset). The results verified the method’s selectivity and applicability for analyzing animal samples from animals treated with Pz-1.

Figure 3.

LC-MS/MS chromatogram of plasma from treated mice, Pz-1-spiked rat plasma (50 nM) and plasma from VC-treated mice. The quantification transition (454.9→315.0 m/z) is displayed for all three chromatograms. Inset: Chromatogram from VC-treated mice at 3×10⁴ times magnification.

4. Conclusions

A rapid, easy-to-use and highly sensitive LC-MS/MS technique was created to measure Pz-1 in rodent plasma. This approach is the first method that has been verified for determining Pz-1 from any biological matrix. The method produced generally excellent figures-of-merit, including low LOD, excellent linearity, excellent selectivity and good accuracy and precision. Moreover, Pz-1 showed remarkable stability in rat plasma. The method was successfully used to analyze Pz-1 from the plasma of IP-treated mice. This method will enable Pz-1 to be further developed as a possible countermeasure for OP poisoning or as a cancer treatment.

Highlights.

• A quick, simple, and highly sensitive LC-MS/MS technique was developed to quantify Pz-1 in rodent plasma.

• This is the first analytical approach that has been proven to measure Pz-1 from any kind of biological matrix.

• The technique yielded outstanding figures-of-merit with good accuracy and precision, low LOD, excellent linearity, and excellent selectivity.