Therapeutic Drug Monitoring of Immunosuppresive

The immunosuppressive agents used to prevent rejection of transplanted organs include cyclosporine (CsA), everolimus (EVE), mycophenolic acid (MPA), prednisolone (PLN), sirolimus (SIR) and tacrolimus (TAC). Because of the narrow therapeutic index and high interand intra-subject variability of these agents, therapeutic drug monitoring (TDM) is an integral part of immunosuppressive therapy following organ transplantation. The immunosuppressants incidence and severity of side effects correlate with the degree of exposure while under-dosed patients can be at a greater risk for allograft rejection. Currently, whole blood or plasma samples that are obtained via venipuncture are used for routine immunosuppressive monitoring. The limitations of venipuncture blood samples include (i) invasive nature associated with the sample collection and (ii) weak correlation with the drug concentration at the site of action. This thesis is consisted of the following sections written in a manuscript format. Manuscript I provides a comprehensive review of literature published on alternative techniques that are proposed to overcome the limitation of venipuncture sampling. These methods include the use of non-conventional techniques, namely, drug monitoring in oral fluids or blood samples obtained from fingertip as well as drug concentration measurement in lymphocytes or transplanted tissue. Drug concentration measurement in lymphocytes or transplanted tissue is primarily aimed at obtaining information on drug level at the site of action thus to facilitate prediction of clinical outcomes. However, these approaches are impractical in clinical setting because of the invasive nature of sampling as well as complicated sample preparation procedures. The objective of finger prick sampling is to mitigate the discomfort and difficulties associated with venipuncture, especially in pediatrics and frail patients. In this approach, the fingertip blood samples are either applied onto a filter paper (dried blood spots) or are processed as a liquid. It has been reported that fingertip sampling was preferred to venipuncture by both patients and healthcare providers. Nevertheless, the main disadvantages of venipuncture whole blood sampling, which is the poor correlation with concentration at the site of action, still exist. Finally, oral fluid sampling is a promising non-invasive method of therapeutic monitoring of immunosuppressive agents. Advances in analytical techniques have enabled measuring drug concentration in minute amount of sample. Drug concentration in oral fluids represents the free fraction which should theoretically represent drug concentration at the site of action. Few comprehensive studies investigated the use of oral fluids as a medium for therapeutic drug monitoring. Therefore, this dissertation is focused on the development of sensitive and robust liquid chromatography tandem mass spectrometry methods for quantification of the most commonly used immunosuppressant agents, tacrolimus and mycophenolic acid. The methods are then used to quantify these agents in oral fluids samples collected from kidney transplant recipients. Manuscript II describes, in details, the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of tacrolimus in oral fluids. This method was validated in accordance with the current Food and Drug Administration (FDA) guideline. The Lower Limit of Quantification of this method is 30 pg/mL that is adequate for measuring tacrolimus concentration in oral fluid samples from transplant recipients. Full separation between tacrolimus and plasma phospholipids components was achieved in very short run time of 2.2 min. Very simple sample predations procedure was followed by extraction 50 μL of oral fluids with 100μL of acetonitrile. Manuscript III in this manuscript, the method presented in manuscript II to quantify tacrolimus in oral fluids. It focused on investigating factors that may affect tacrolimus measurement in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after give a saliva stimulant), sampling time, and blood contamination expressed as salivary transferrin level. The correlation between tacrolimus concentration in blood and oral fluids was investigated under these conditions. Correlation analysis revealed that samples collected after mouth rinse and at fasting provided better correlation in tacrolimus concentrations in blood and oral fluid. Manuscript IV: Liquid chromatography tandem mass spectrometry methods was developed and validated according to current FDA Guidelines to quantify mycophenolic acid and its glucuronide metabolites in oral fluids, total concentration in plasma, and unbound fraction in plasma. Full separation of mycophenolic acid, metabolites, and plasma phospholipids was achieved within the total run time of 2.8 min. Manuscript V: The assay described in manuscript IV was used to quantify mycophenolic acid and glucuronide metabolites in oral fluids. The aim was to investigate factors that may affect mycophenolic acid and glucuronide metabolites concentration in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after saliva stimulation), sampling time, and blood contamination expressed as salivary transferrin level. The result of this study indicated that the blood contamination had an insignificant effect on the concentration of mycophenolic acid and metabolites in oral fluids. In addition, a good correlation was observed between AUC0-12 of MPA in OF samples and unbound and total MPA. In contrast, a weak association was observed between MPAG concentrations in oral fluids with total and unbound plasma concentration. Manuscript VI: PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. In this manuscript, the development and validation of a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain was described using liquid chromatography-tandem mass spectrometry. Full separation was achieved between the analyte and phospholipids of the three matrices within the total chromatographic run time of 2.2 minutes. The manuscript also identified and described the abundance of phospholipids contents of the three matrices. The developed method successfully used to quantify the analytes in the three matrices as part of pre-clinical and ongoing clinical studies.

setting because of the invasive nature of sampling as well as complicated sample preparation procedures.
The objective of finger prick sampling is to mitigate the discomfort and difficulties associated with venipuncture, especially in pediatrics and frail patients. In this approach, the fingertip blood samples are either applied onto a filter paper (dried blood spots) or are processed as a liquid. It has been reported that fingertip sampling was preferred to venipuncture by both patients and healthcare providers. Nevertheless, the main disadvantages of venipuncture whole blood sampling, which is the poor correlation with concentration at the site of action, still exist. Very simple sample predations procedure was followed by extraction 50 µL of oral fluids with 100µL of acetonitrile.
Manuscript III in this manuscript, the method presented in manuscript II to quantify tacrolimus in oral fluids. It focused on investigating factors that may affect tacrolimus measurement in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after give a saliva stimulant), sampling time, and blood contamination expressed as salivary transferrin level. The correlation between tacrolimus concentration in blood and oral fluids was investigated under these conditions. Correlation analysis revealed that samples collected after mouth rinse and at fasting provided better correlation in tacrolimus concentrations in blood and oral fluid.
Manuscript IV: Liquid chromatography tandem mass spectrometry methods was developed and validated according to current FDA Guidelines to quantify mycophenolic acid and its glucuronide metabolites in oral fluids, total concentration in plasma, and unbound fraction in plasma. Full separation of mycophenolic acid, metabolites, and plasma phospholipids was achieved within the total run time of 2.8 min.
Manuscript V: The assay described in manuscript IV was used to quantify mycophenolic acid and glucuronide metabolites in oral fluids. The aim was to investigate factors that may affect mycophenolic acid and glucuronide metabolites concentration in oral fluid, namely, sampling condition (resting, after mouth rinsing, and after saliva stimulation), sampling time, and blood contamination expressed as salivary transferrin level. The result of this study indicated that the blood contamination had an insignificant effect on the concentration of mycophenolic acid and metabolites in oral fluids. In addition, a good correlation was observed between AUC 0-12 of MPA in OF samples and unbound and total MPA. In contrast, a weak association was observed between MPAG concentrations in oral fluids with total and unbound plasma concentration.
Manuscript VI: PF-5190457 is a ghrelin receptor inverse agonist that is currently undergoing clinical development for the treatment of alcoholism. In this manuscript, the development and validation of a simple and sensitive assay for quantitative analysis of PF-5190457 in human or rat plasma and rat brain was described using liquid chromatography-tandem mass spectrometry. Full separation was achieved between the analyte and phospholipids of the three matrices within the total chromatographic run time of 2.2 minutes. The manuscript also identified and described the abundance of phospholipids contents of the three matrices. The  recovery and absolute matrix effect, expressed as mean peak area ± std ( n =3) 80 Table 3

Introduction
Therapeutic drug monitoring (TDM) is an integral part of immunosuppressive therapy following organ transplantation because of the narrow therapeutic index and high inter-and intra-subject variability of these agents [1][2][3][4]. The immunosuppressive agents used in solid organ transplant include cyclosporine (CsA), everolimus (EVE), mycophenolic acid (MPA), prednisolone (PLN), sirolimus (SIR) and tacrolimus (TAC) [5]. The incidence and severity of side effects of immunosuppressant agents correlate with a high exposure [5], while under-dosed patients can be at a greater risk for allograft rejection [1,5]. Currently, whole blood or plasma samples obtained through venipuncture are used for routine immunosuppressive monitoring [5]. The limitations of venipuncture blood samples include the invasive nature associated with the sample collection and the weak correlation with the drug concentration at the site of action. In this review, these limitations and proposed alternative methods will be discussed.

Use of tandem mass spectrometry (LC-MS/MS) in drug monitoring
Advances in LC-MS/MS have enabled researchers to measure drug concentrations in limited sample volumes with adequate sensitivity, selectivity and robustness. This review will focus mainly on the use of LC-MS/MS in immunosuppressive agents in TDM using alternative matrixes, namely oral fluids (OF), dried blood spots (DBS), peripheral blood mononuclear cells (PBMC), and a biopsy sample from the implanted organ. Other techniques, such as high-performance liquid chromatography (HPLC) and immunoassays, will be briefly discussed wherever significant findings have been reported.
The use of LC-MS/MS has long been a gold standard in pharmacokinetic studies [6], and it is becoming an increasingly used technique in clinical laboratories [7]. A reduced chromatographic run time and increased sensitivity are typically achieved using ultra-performance liquid chromatography (UPLC) and newer stationary phases [8,9]. LC-MS/MS has enabled researchers to quantify lower drug concentrations in small blood sample volumes (i.e., 4-10 µL) [10][11][12][13][14][15] with higher specificity in comparison with immunoassays [16][17][18][19][20]. In addition, LC-MS/MS allows the simultaneous quantification of more than one analyte and/or metabolite [9,21] with different physiochemical properties with a high degree of sensitivity and selectivity [22].
LC-MS/MS is a system that combines high-performance chromatography (HPLC) with mass spectrometry (MS). Three atmospheric pressure ionization (API) techniques, namely electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and atmospheric-pressure photo-ionization (APPI), are typically employed [23]. These techniques provide highly precise quantitative analysis with minimal sample preparation of complex samples such as blood, plasma and OF [22,24,25]. ESI technique, most commonly used in quantifying polar to ionic compounds, and in metabolomics and proteomics studies [23]. The main challenge that may hinder the LC-MS/MS method development is the matrix effect (ME), which may produce erroneous results [26,27]. Proper cleanup of samples [26], the use of a deuterated internal standard [21], and chromatographic separation of analytes from regions of ion enhancement or suppression can mitigate/eliminate the effect of ME [28].

Oral fluids as a matrix for therapeutic drug monitoring
Oral fluids have been a subject of interest as an alternative medium to venipuncture blood [24,25,[29][30][31][32][33][34][35][36][37][38][39]. The main advantage of OF sampling is the noninvasive sample collection, permitting more frequent sampling [40] and allowing more convenient selfsampling [41]. Moreover, OF sampling offers a significantly lower cost per sample [41,42]. In addition, the drug portion measured in the OF represents the free drug concentration [41,42] (Figure 2). Given that the free drug concentration is responsible for the pharmacological and toxicological effects [4,43,44], measurement of the drug concentrations in OF may provide a better prediction of clinical outcomes and toxicity [34,45]. Therefore, salivary drug level measurements are much easier and faster than quantifying the free drug concentration in plasma [25,38].
Drugs enter the OF mainly via passive diffusion [35]. Thus, physiochemical properties, including protein binding, ionization, lipophilicity, and molecular weight, are important determinants for the entry of a drug into the OF [35,45]. The ability of a drug to diffuse and equilibrate between the plasma and tissues is governed by its free fraction [ [35,45,46]. According to Lipinski's rule of five, a molecular weight < 500 is  [29,34,35,45,46]. The degree of ionization of a drug is determined by its pKa (the pH at which 50% of the drug is found in ionized form) and the pH of the medium [33]. Theoretically, basic drugs with pKa values less than 5.5 and acidic drugs with pKa values greater than 8.5 are not affected by changes in salivary pH (5.8-7.8) [45,48]. Under these conditions, drugs predominantly exist in unionized form, therefore; they have higher lipophilicity and consequently cross biological membranes more easily [29,35]. The chemical structure and physicochemical properties of immunosuppressive agents are presented in Figure   1 and Table 1 (Table 1), and therefore high permeability is predicted despite the low free fraction. A low free fraction thus will be the rate-limiting factor for penetration of drug into saliva making saliva a suitable specimen to measure the unbound concentration of immunosuppressive agents.

Resting vs. stimulated OF sampling
The concentrations of certain drugs in the OF are affected by the salivary flow rate [29,35,48]. Stimulated OF has less contact time in comparison to resting OF, consequently reducing the influence of tubular re-absorption and secretion [29,48].
Stimulation may alter the salivary composition and pH [51], thereby may affect the partitioning of drugs between the OF and plasma [52] by modifying the ionized portion. Changing the salivary flow rate alters the correlation between the plasma and OF drug concentrations of some drugs but has little to no effect on others [29,48].
Acidic drugs mainly exist in non-ionized forms at a lower salivary pH, which allows better correlation with the plasma concentration [33]. In contrast, basic drugs tend to accumulate in acidic saliva because they exist predominately in the ionized form, which limits their movement across biological membranes [29] (Table 1) In addition, food stimulates protein-rich OF, compared with other stimuli that produce protein-poor OF [33]. No published studies have investigated the effects of salivary stimulation on the distribution of immunosuppressive agents into the OF. consisting of cotton, polyester or polyethylene roll were highly rated by patients and investigators based on their ease of use and practicality. The OF collection methods used in the immunosuppressive agent quantification assays are shown in Table 2.
The adsorption of TAC into plastic materials, including polyolefin and polyvinyl chloride used in making central venous catheters, has been reported [54]. However, a recent study showed that the stability of TAC was not compromised when it was stored in either glass or plastic containers [24]. The yield of TAC obtained from OF samples with passive drool and polypropylene Salivette® devices was also studied. A modest correlation (r = 0.57) was reported in TAC concentrations in drool and Salivette® samples [24]. Although minimal to no interaction was observed between CsA and plastic/glass materials used in the manufacture of blood collection tubes, the adsorption of CsA into peripheral and indwelling catheter sites has been reported [55].
To prevent non-specific binding and to minimize the risk of adsorption, siliconization (i.e., the application of a thin layer of highly hydrophilic material) of the OF collection and storage containers may prove to be beneficial [39].

Sample preparation and extraction
The mucopolysaccharide content of OF may interfere with the accuracy of pipetting [58]. Sample homogenization aids in breaking down salivary proteins and improving extraction yields [38]. Subjecting OF samples to freeze and thaw cycles followed by centrifugation facilitates sample processing and breaks down mucopolysaccharides [58]. Simple pre-analysis treatment and protein precipitation using 2-3 volumes of acetonitrile (ACN) has been shown to provide sufficient sample cleanup and good recovery [24][25][26]. Some methods employ more labor-intensive techniques, including SPE and drying for sample cleanup [37-39].

Blood contamination of oral fluid
Predicting the effect of mouth injuries based on the concentration of endogenous concentration. In another study [24], the influence of salivary blood contamination on the TAC level was investigated. When 1 mL of blank OF samples spiked with different volume of blood (<1, 2, 5, and 10 µL) contained TAC (11.2 μg/L) were analyzed, only samples that were spiked with 2, 5, and 10 µL of TAC displayed visual signs of blood contamination together with proportional increases in TAC concentrations up to 28%. Thus, visual inspection might be sufficient for sample exclusion due to blood contamination for TAC.

Measurement of immunosuppressive agents in oral fluids
In the following paragraphs, the physiochemical characteristics of immunosuppressive drugs will be presented, and LC-MS/MS methods that utilize OF will be discussed.

Cyclosporine
Cyclosporine is an extremely lipophilic compound that is mostly distributed in plasma

Tacrolimus
Tacrolimus is a highly lipophilic compound ( Table 2) with a plasma free fraction of approximately 1% [3]. The unbound fraction is significantly affected by changes in plasma lipoprotein concentrations after liver transplantation [43], which may lead to incidences of rejection and/or toxicity [43,63]. There is only one published method for the utilization of the OF matrix for TAC quantification [24] ( Table 2).

Mycophenolic acid
The unbound fraction of MPA ranges from 1 to 2.5% [4]. In patients with severe renal impairment, the concentration of the major MPA metabolite, MPA-glucuronide (MPAG), may increase up to 3-6-fold. This increase in MPAG leads to displacement of MPA from its binding sites [4], and as a result, the MPA-free fraction may increase up to 7% [4]. Mycophenolic acid has a low molecular weight and lipophilic nature (logD 0.76 at pH 7.4) (  Table 2).
Some studies [24,39] have focused on finding an association between total drug concentrations in the blood and OF. Because the drug fraction in OF theoretically represents the unbound portion, a good association with the free fraction in the blood should be pursued. The total drug concentration may not correlate very well with the free fraction [4,43]. However, OF sampling may be considered a non-invasive alternative to venous blood sampling if a good correlation between total blood and OF drug concentrations is established. collecting device from the fingertip or venipuncture is pipetted onto a predetermined circular area of a special filter paper [15,77,83]. The latter approach guarantees the application of a precise amount of blood sample to the filter paper. However, this additional step may make home self-sampling less appealing [15]. In addition, capillary self-sampling may result in a significantly different result from sample collection by healthcare professionals [15]. Liquid fingerprick blood sampling involves the direct extraction of blood samples in liquid form, which are collected using EDTA-containing devices such as Microvette™ [12,15] or Microtainer™ tubes [13].

Extraction procedure and recovery of DBS and LFB sampling
A disc of the blood spot with a diameter between 4 and 8 mm is removed using a special puncher. The sample extraction ranges from simple vortex mixing [15,75,82

Effect of the punching location
The distribution of analytes may differ between the center and the outer area of the This utilizes simple and practical procedures using volumetric absorptive microsampler devise (VAMS). It consists of porous absorbent polymeric tip capable of absorbing more precisely 10 µL of blood utilizing capillary force.

Matrix effect
The extracted matrix from DBS appears to have a negligible effect on ME

Stability
Despite the use of the same DBS collection paper (Whatman 903), a discrepancy in stability has been reported, especially for CsA ( Table 3). Leichtle  (see Table 3).
Tacrolimus that was measured in EDTA venous blood (50 µL) applied immediately onto a filter paper and dried at room temperature for 3 hours showed stability for up to

Patient preference
Self-fingerprick sampling is well tolerated with no serious discomfort as reported by children [14,81] or adult transplant patients [12,79,95]. In solid organ transplant patients, LFB was preferred (60%) over venipuncture sampling, and approximately 68% of patients favored the use of DBS over LFP sampling (18%) [15]. The sampling process for LFB may be troublesome for some patients and therefore may produce poor sampling [12,15]. Nonetheless, unsupervised capillary and DBS self-sampling can be improved by providing brief instructions or over-the-phone consultation [12,73].

Clinical application of DBS and LFB
The mean difference in CsA concentrations is significantly higher in DBS prepared from capillary tube-collected fingertip blood than from venous blood at C 0 and C 2 [15]. Despite the low recovery of EVE from DBS (76.5%), the concentration of EVE in DBS was slightly higher than in venous blood. A high correlation between venous and fingertip samples is expected because both represent whole blood. However, a statistically significant higher TAC has been reported in LFB samples compared to venous blood, but the mean difference was clinically insignificant (0.29 ng/mL, 95% CI 0.09-0.49), and a good correlation was reported (r 2 =0.845) [14]. In contrast, the CsA venous blood level was statistically significantly higher than in LFB [11]. The mean difference was 9.5 ng/mL (95% CI 0.8-18.2 µg/L, P<0.03), however, a strong association was also reported between venous and LFB samples (r 2 = 0.96, P < 0.001).
Because fingertip sampling utilizes whole blood, a lack of correlation is expected between the obtained levels of immunosuppressive agents in DBS or LFB and their levels at the site of action (see sections 3 and 4). However, the relative ease of DBS and LFB sampling compared to venipuncture, the possibility of home self-sampling, and the stability during storage and transportation suggest that both of these techniques have the potential to replace venipuncture in TDM.  Table 5).

Intracellular concentration
There is a histologically and clinically proven rejection associated with a lower level of TAC in PBMCs measured at day 7 post-transplantation in liver transplant recipients [117].

Sample preparation and extraction of immunosuppressants from PBMCs
The volume of whole blood needed to prepare PMBCs ranged from as low as 1.5 mL to as high as 10 mL (Table 5). To prevent immunosuppressant efflux from PBMCs during sample preparation, it is crucial to add a P-gp inhibitor such as verapamil or to perform the preparation procedures at 4 °C. The main limitations of intracellular drug concentration quantification methods the invasive nature of obtaining blood samples and the labor-intensive sample preparation procedures, which involve cells counts, drying and reconstitution and solid-phase extraction.

Intra-tissue concentration
Early reports on the measurement of intra-tissue concentrations of immunosuppressive  Table 6).
Post-mortem examinations have revealed that CsA and its metabolites accumulate rapidly in tissues after administration [133]. Measured using HPLC, the total concentration of CsA and its metabolites reached levels that were 53-fold higher in organs and tissues than in whole blood [133]. Tissue CsA concentrations were highest in the pancreas, followed by the spleen, liver, kidney, lung, and heart. In a recent study

Effect of ABCB1 gene polymorphisms on tissues concentrations of immunosuppressive agents
The inter-subject variability of P-gp substrates in tissues may be the result of genetic polymorphisms in P-gp transporters. Indeed, significantly higher TAC concentrations have been found in hepatic tissue from patients carrying alleles with reduced activity [140]. There were significantly higher hepatic tissue TAC concentrations, expressed as the geometric mean of the dose-normalized hepatic concentration, in carriers of the reduced-activity 1199A allele (1199A) than in non-carriers (P=0.036).
Correspondingly, hepatic tissue obtained from carriers of the 236C>T and 2677G>T/A alleles demonstrated a higher TAC concentration, expressed as the geometric mean of the hepatic concentration (P = 0.014 and 0.035, respectively). Finally, although CYP3A-metabolizing enzymes are expressed in hepatic tissues, they have no effect on hepatocyte TAC concentrations [140]. In summary, the blood concentration of immunosuppressive drugs in solid organ transplant recipients is a poor predictor of intra-hepatocyte levels.

Conclusions and future prospective
Optimal exposure to immunosuppressant agents is required to improve allograft survival and reduce toxicity. Despite its limitations, venous blood remains the    was used to control the system and data acquisition, and data processed using TargetLynx™ tool. The UPLC system had a binary pump and equipped with built-in column heater. Twenty micro-litters sample loop was used to deliver 10 µL of the samples in partial loop mode. For salivary blood contamination assay, SpectraMax M5e Microplate Reader (Sunnyvale, CA, USA) was used.

Sample extraction
Calibration standards, quality controls (QCs), blank, and patients' OF samples were allowed to thaw at room temperature. After vortexing for 5 seconds, samples were

Sensitivity and selectivity
Lower limit of quantification (LLOQ) was set at the concentration with a signal to noise ratio (S/N) of at least 10, accuracy between 80-120%, and Coefficient of Variation (CV) less than 20%. Acceptance criteria for QCs were accuracy between 85-115% and CV less than 15%. Selectivity was assessed by inspecting the presence of noise or peaks in chromatograms that represented blank OF samples (from 6 donors) as compared with LLOQ sample chromatogram.

Stability
Stability studies were performed by measuring TAC concentrations in QC1 and QC3, in three replicate. Freeze and thaw (after three freeze and thaw cycles), bench-top, auto-sampler (by re-injecting one of validation batch after it was left in the auto-sampler for 24 hr and 48 hr), and short-term stability up to one month were investigated.

Matrix effect and Recovery
The presence and possible matrix effect (ME) in OF studied in two different ways. In

Results and discussion
Recommended TAC C 0 therapeutic blood concentration in kidney transplant recipients is between 15-20 µg/L immediately after transplantation [3]. TAC dose is tapered gradually, and the maintenance C 0 can be as low as 5-7 µg/L after first year posttransplantation [3]. Since only 1% of TAC amount found in the unbound form that is capable of reaching the OF, the expected OF concentration would range between 0.050-200 ng/L. Therefore, highly optimized mass spectrometry and chromatographic conditions were sought to develop a method with adequate selectivity and sensitivity.
To achieve the highest selectivity feasible, different columns were tested. Acquity UPLC BEH C18 seemed to be a good choice as it gave sharp and symmetric peaks.
Given the above-mentioned UPLC and mass spectrometry conditions, it was possible to set LLOQ at 10 ng/L with signal/noise ratio of more than 10 ( Figure 1A). No carryover was detected when a double blank OF sample was injected following highest calibration concentration ( Figure 1B). The calibration curve was constructed by plotting nominal standards concentration against peak area ratios of the analyte to IS and fitted with 1/x weighted least squares linear regression. The method demonstrated adequate accuracy and precision with QCs accuracy between 94.5-103.6%, and CV within 4-9.8 ( Table 1). The correlation coefficients (r 2 ) calculated from validation batches (n=3) were between 0.998-0999.
Stability studies, namely, freeze and thaw, bench top, auto-sampler, and short-term storage at -80 °C for up to four weeks were conducted ( Table 2). No stability problems were noticed, and TAC was stable in extracted matrix for up to 48 hrs.
Possible interference from endogenous substances in OF was investigated.
Chromatograms obtained from acquiring a pooled blank OF from six donors ( Figure   1B) and blank neat solution (66% ACN) ( Figure 1C). No signs of interference were noticed.
Using methanol instead of ACN as organic solvent helped improving the sensitivity,  (Figure 3). The other two phospholipids that have m/z > 700 were less problematic and eluted way after analytes of interest.
In total 181 samples collected from 71 kidneys transplant patients analyzed only one sample had concentration lower than LLOQ with calculated concentration around 8.5 ng/L, collected 2 hr after dose, even with the corresponding blood concentration was within the normal range (11.8 µg/L). The concentration of TAC ranged from 11.7-76 2864.4 ng/L and 1.7-46.06 µg/L for OF and whole blood, respectively. The clinical finding of this study will be presented in a separate manuscript.

Incurred sample re-analysis
The incurred samples reanalysis test was performed by re-analyzing about 10% of the samples (19 samples) [146]. Whenever many samples were available per patient, two samples were selected to represent absorption and elimination phases. The difference between the paired measurements were normally distributed, therefore, the use of Bland-Altman method was justified [151]. Repeatability was tested visually ( Figure 3) and statistically. Good agreement between the two repeated measurements can be observed in Figure 3, which plots the percent differences between paired repeated measurements against their mean. All points lie between or near the 95% confident interval lines. The 95% limit of the agreement was from -19.16 to 31.98. The bias (mean the difference between two occasions) was 6.40.

Conclusion
In this paper, development and validation of a very sensitive, selective and robust method is presented. Simple sample preparation and extraction protocol was developed and used to provide minimum sample dilution and appropriate samples cleanliness, excellent recovery and minimum sample components interference. In addition to lowest reported LLOQ of TAC, this work is the first to study the effect of different proportions of precipitating solvent (ACN) on the ME and recovery. In addition, this is the first report that investigated and described phospholipids 77 chromatographic elution behavior and the possible interference of phospholipids with the analyte in the OF.    Table 2

Introduction
To prevent allograft rejection, organ transplant recipients require chronic immunosuppressive therapy [1]. Tacrolimus (TAC) is a widely prescribed immunosuppressive agent for solid organ transplant recipients [1]. It acts by binding to an immunophilin, FK506-binding protein 12 (FKBP12) [2]. The complex then inhibits calcineurin phosphatase and thereby halts T-cell activation [3]. TAC is highly lipophilic and excreted from the body after undergoing extensive metabolism by the CYP450 3A4/5 enzymes [4]. Bioavailability of TAC varies significantly due to genetic polymorphism in CYP3A as well as co-administration of CYP3A enzymes inhibitors or inducers [5][6][7][8], thus increasing intra and inter-subject variability in pharmacokinetics [9][10][11][12]. In addition, TAC is a substrate for glycoprotein efflux transporter (P-gp), which is also known as multidrug resistance protein 1 (MDR1) encoded by the ABCB1 gene [13]. Differences in the expression of MDR1 [13] and genotype [14] may contribute to inter-individual variability in tacrolimus pharmacokinetics. Given narrow therapeutic index and high variability, ongoing therapeutic drug monitoring is essential to maintain allograft survival and reduce toxicity [15].
Venipuncture is the recommended medium for TAC therapeutic drug monitoring [1].
However, due the invasive nature of blood sampling, alternative matrices were investigated including dried blood spot and oral fluid (OF) monitoring [16][17][18][19][20]. Oral fluid has attracted great attention as an alternative medium to venipuncture blood [21][22][23]. The main advantage of OF sampling is noninvasive sample collection, significantly reduced sample collection cost [24,25] and the possibility of home self-sampling for patient convenience [24]. In addition, biological barriers are permeable to free drug fraction only considering that protein bound complexes are excluded from passive diffusion because of their large size [26]. Consequently, the portion of a drug that is present in OF represents the unbound fraction [26]. Given that the free fraction is responsible for therapeutic effect and toxicity [27], measuring drug concentration in OF may give better prediction of therapeutic outcomes.
The area under the concentration-time curve (AUC) and maximum concentration (C max ) correlate better with clinical outcomes and toxicity that blood sample [28].
Since calculation of AUC requires collection of several samples over a 12 hour hour dosing interval, venipuncture blood sampling is impractical for routine calculation of AUC. The simplicity of OF sample collection allows multiple sampling, therefore, estimating AUC and C max will be possible. A few reports were published on immunosuppressant, namely, cyclosporine A [19,29], mycophenolic acid [17,30,31] and TAC [20].
The aim of this study was to study factors that may affect correlation between the TAC concentrations in blood and OF, as well as the quality of OF samples obtained at different sampling condition.

Study population
Studies protocols approved by Institutional Review Board of Rhode Island Hospital (Providence, RI). Samples included in this paper were collected, in two studies, from patients attending kidney transplant clinics. Recruited patients were on triple immunosuppressants regimen included tacrolimus, prednisone, and mycophenolic acid.

Patients Samples
On the study day, patients underwent the physical examination by the physician and asked to sign the informed consent. In the first study, venous blood samples (about 4 mL) were collected in ethylenediaminetetraacetic acid (EDTA) accompanied by passive drooled rested OF samples collected in siliconized plastic cups. Samples collected intermittently at certain time points, including pre-dose (time 0 = C 0 ). In the second study, blood samples were collected at C 0 and C 2 . Matching OF samples collected with ± 5 min from blood sample time at resting, 5 min after mouth rinsing using bottled water, and instantly after taking a saliva stimulant (patients asked to put a commercial sour candy in their mouth for 10 second with continues tong movement). After pre-dose samples collection, patients were given a voucher for free breakfast and asked to report back at study location shortly before C 2 sampling time when blood and corresponding OF samples were collected. All blood and OF samples kept on dry ice till transferred to the Biomedical and Pharmaceutical Sciences (BPS) department at University of Rhode Island and stored at -80 °C till analyzed. Table 1.

Measuring TAC in blood and OF
Details of the LC-MS/MS method used to quantify TAC blood concentration is described elsewhere [32]. A simple sample preparation and extraction procedures were followed, involved adding 50µL of OF sample with 100µL of ACN precipitating solvent containing internal standard (ascomycin, 600 ng/L) in 1.5 mL polypropylene tube. After vortex mix for 10 seconds, the mixture was centrifuged at 10,000xg for 5 min at 20°C. The supernatant was then transferred into an auto-sampler vial, and 10 μL was injected.
The dynamic range of was 30-4800 ng/L. The lower limit of quantification (LLOQ) was set at the concentration that had a signal-to-noise ratio (S/N) of ≥10;; accuracy of 80-120%; and a Coefficient of Variation (CV) less than 20%. Acceptance criteria for QCs included accuracy between 85-115% and CV less than 15%. Selectivity assessed by inspecting the presence of noise or peaks in chromatograms represent blank OF samples injections (from 6 donors) compared with LLOQ sample chromatogram.

Statistical data analysis
Statistical analysis was performed using the SPSS software (version 22, SPSS Inc., Chicago, IL, USA). Normal distribution of the data was checked graphically and confirmed with the Shapiro-Wilk test, and nonparametric tests were used whenever needed.

Genomic studies:
DNAzol kit was used to extract genomic DNA from blood samples obtained from

Salivary blood contamination
To assess and quantify possible salivary blood contamination, transferrin kit from Salimetrics LLC (State College, PA, USA) was used following manufacture's recommendation [33]. Transferrin quantification was performed using SpectraMax M5e Microplate Reader (Sunnyvale, CA, USA).

12-hours profile
Eighty-five OF samples collected from 10 patients at rest. All samples had TACs within assay's validated range (30-4800 pg/mL). The concentration of TACs and blood were 5.57±2.58 and 863±641, respectively 2 hours profile study

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In total, 184 OF samples were analyzed. Five samples were excluded (four samples had TAC concentration less than LLOQ all of them were stimulated samples, and one rested C 0 sample had visible blood contamination). On the other hand, 4 OF samples had TAC concentration higher than upper limit of quantification; all of them had salivary transferrin (TRNs) level higher than transferrin salivary assay quantification range (0.08 -6.6 mg/dL). Four additional samples had TRNs higher than the upper limit of quantification. In total 171 samples were eligible for further data analysis.
Following manufacturer's recommended threshold of ≤1mg/dL TRNs, 131samples would be eligible for further analysis ( Table 2). Samples with TRNs level >1mg/dL were 14 rested, 11 rinsed and 4 stimulated samples collected at C 0 and 7 rested, 2 rinsed and 2 stimulated samples collected at C 2 .

Blood contamination and TACs concentration
Transferrin is a plasma protein with molecular weight of 76000 [33]. The presence of TRN in OF is an indication of injury in oral cavity [33]. The possibility of salivary blood contamination may increase in the presence of micro injuries from poor oral hygiene, some infectious diseases and smoking [33]. TRNs level showed to have diurnal variation, with a higher level in the afternoon compared with earlier collected samples [34]. Contradicting finding regarding the effect of gender differences in the TRNs level in children (higher in boys) [34] and adults (higher in females) [35].
Therefore, using TRNs as biomarker of salivary blood contamination should take into the consideration the physiological and environmental factors that may alter the TRNs level. About 85% of tacrolimus distribute into red blood cells [36]. Therefore TRNs >1 mg/dL), in pre-dose fasted samples [17]. In this study, we measured the transferrin concentration in oral fluid as a biomarker for blood contamination to determine the threshold value at which TACs measurements would be compromised.
Following manufacturer's recommended threshold of ≤1mg/dL TRNs, data analysis revealed a significant correlation between TACs and TRNs concentration (p-values <0.05) in some sampling condition ( Table 2, R-values denoted with *).
High TRNs is an indication of salivary blood contamination that may artificially overestimate drugs concentration in OF [34]. Assumed increases of TACs levels in response to high TRNs level was investigated in all sub-groups by calculating mean+1STD and mean+2STD of TRNs in all samples with TRN level within the dynamic range of the assay ≤6.6mg/dL ( Table 3). All samples that have TRNs level falls within mean+1STD or mean+2STD were included, and the correlation between produced values (mean+1STD or mean+2STD) and TACs was tested. As can be seen from  Table 6) plot against TACs, no correlation is seen (Figures 1). These results are agreement with previous study [20]. In this study, the effect of blood contamination on TACs level was investigated by spiking OF with increasing amount of blood contained 11.2 μg/L TAC. Only samples that showed signs of discoloration had elevated TAC level between 4.5 and 28%. Given all above, it seems that there is insignificant/weak correlation between TACs and TRNs concentration in samples with TRNs ≤6.6mg/dL;; therefore all samples with TRNs ≤ 6.6mg/dL were included in further data analysis.

Effect of different sampling conditions
Changing salivary flow rate may alter the drug concentration drug concentrations in the OF via altering contact time and the pH, consequently, affecting tubular reabsorption and secretion [21,38]. Changes of flow rate may affect some drugs but has little to no effect on others [21,38]. Tacrolimus is a highly lipophilic compound with logP and pka value of 3.19-5.59 and 9.96, respectively [39]. These characteristics make TAC non-ionized in physiological pH, therefore, ideal for OF therapeutic drug monitoring. Additionally, food consumption produces protein-rich OF compared with protein-poor OF produced from other stimuli [40]. In this study, the effect of different sampling conditions on quality of OF samples, as determined by TRNs level, and the correlation between TAC concentrations in OF and blood, were studied.

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The concentrations of TAC in OF in all subgroups are showed in Figures 2A and 2B.
In both time points, there is as decrease in concentration, with the highest level in the rested sample followed by rinsed and stimulated samples.

Correlation between TAC in OF and blood
Interestingly, when comparing same sampling conditions across the two time points, the mean concentration of TACs in C 2 samples were always lower compared to C 0 samples (Figures 3A, 3B and 3C, not statistically significant), despite the fact of significantly higher TAC in C 2 blood samples ( Figures 3D). We attribute this to the possible effect of food as C 2 samples were collected after serving the breakfast.
Correlation between salivary and blood TAC concentrations are presented in Figures   4A and 4B. As can be seen, the correlation was best in rinsed samples collected at C 0 ; therefore, this subset of data was selected to check possible covariate effect.
Metabolizing enzymes CYP3A4 [41] and P-gp transporter are expressed in minor and major salivary glands [42,43]. The possible effect of genetic polymorphism in CYPA3 enzymes and P-gp on the association between TAC concentration in OF and blood was examined. Nonetheless, no statistically significant differences were seen in different genotyped patients.

hours profile study
The High correlation between TACs and TRNs was seen in samples collected over 12 hours period (Figure 5A, R= 0.67, p <0.001). However, weak correlation was seen between TAC concentration in OF and blood ( Figure 5B, R = 0.13, p= 0.21). These results are online with these obtained in 2 hours profile study in which poor correlation in TAC concentration in OF and blood was seen.

Conclusion
Using OF as an alternative to blood for TDM is appealing due to ease and low-cost of sampling. Many factors may alter drugs levels in OF. Results of this study indicate that salivary blood contamination has minimal on TACs when TRN level was ≤ 6.6 mg/dL. Better correlation between oral fluid and blood tacrolimus concentration was observed in fasted samples collected at pre-dose as compared with non-fasted samples collected post dose.       (AcMPAG) [2]. MPA highly binds to plasma protein with only 1-3% found in free form [2]. In patients with compromised renal, MPAG metabolites level may increases by 3-6 folds, resulting displacement of MPA from plasma protein binding sites [2]. As a result, MPA free fraction may increase up to 7% [2]. Currently, whole blood or plasma obtained through venipuncture is used for TDM of immunosuppressive agents [3]. Because of the invasive nature of blood sampling, alternative matrices were investigated, including OF [4][5][6] and dried blood spot [7]. Because of the narrow therapeutic index, therapeutic drug monitoring of MPA is recommended.

Statistical data analysis
Statistical analysis was performed using GraphPad Prism software.

Sensitivity and selectivity
The method was validation in accordance with the current version of FDA guidance for industry bioanalytical method validation [9]. The lower limit of quantification (LLOQ) was determined by the concentration that had signal/noise ratio of at least 10, accuracy between 80-120%, and Coefficient of Variation (CV) less than 20%.
Acceptance criteria for QCs were accuracy between 85-115% and CV less than 15%.
Selectivity assessed by inspecting the presence of noise or peaks in chromatograms represent blank OF, whole plasma and plasma ultrafiltrate samples (from 6 donors) compared with LLOQ samples chromatograms.

Stability
Stability studies were performed by measuring MPA and MPAG in each matrix, in three replicate, at QC1 and QC3 concentrations. Stability studies included freeze and thaw (after three freeze and thaw cycles), bench-top (for up to 8hrs) and auto-sampler (by re-injecting one of validation batch after it was left in the auto-sampler for 24hrs).

Matrix effect and Recovery
The presence and the possible effect of matrix effect (ME) in all matrices studied in two different ways.

Sensitivity and selectivity
In-source conversion of glucuronide metabolites MPAG to MPA has been reported [10], therefore full chromatographic separation is essential to avoid over estimation of  Figure 2.
Calibration curves ranges of the anlytes were 0.001-1µg/mL and 0.004-1µg/mL in OF; 0.05-50 µg/mL and 1-100µg/mL in plasma ultrafiltrate; and 0.1-151µg/mL and 1-100 µg/mL in plasma; for MPA and MPAG, respectively. Analytes to internal standard peak ratio against nominal concentration used to construct the calibration curve and fitted using (1/x) weighting method. To determine accuracy and precision of the assay, three different batches of OF and plasma (for total and unbound concentration) were spiked with working stocks solution to achieve QCs concentrations (6 replicate) and extracted as described in sample extraction section. Accuracy and precision of the assay are showed in Table 1.

Stability
Bench top, freeze and thaw, auto-sampler, were studied ( Table 2). No stability problems were noticed, and analytes were stable in extracted matrices for up to 24hrs.

Recovery and matrix effect
Samples processing and extraction procedures showed excellent recovery form both OF and plasma. The recovery ranged in OF and plasma from 88.71-103.09% for both MPA and MPAG (Table 2). In a recent paper [4] MPA and its metabolites, MPAG, were quantified simultaneously with 82.1and 65.7% recovery, respectively, following solid phase extraction procedures.
Biological fluids contain endogenous components that may interfere and compete with analytes of interest at the ionization site in LC-MS/MS [11][12][13]. The ME is the term that describes this phenomenon. The ME may lead to either ionization suppression or enhancement, both of which may compromise the integrity of the results [11][12][13].
To investigate possible inference of matrices component, post-column infusion technique was utilized [13]. Additionally, ME was also investigated visually by monitoring their MRM transitions.
Finally, the use of plasma samples obtained from healthy volunteers in preparing calibration curve may not completely mimic plasma obtained from transplant patients.
Transplant patients usually co-prescribed a large number of medications to prevent rejection and manage coexisting conditions [14,15]. Therefore, incurred sample reanalysis test was performed by re-measure about 10% of patient's samples [16]. As can be seen in Figures 5A, 5B, and 5C, great agreements between two repeated measurements of MPA (upper) and MPAG (lower) in OF, plasma, and plasma ultrafiltrate, respectively. In these figures, Bland and Altman plots constructed by plotting the differences between paired repeated measurements against their average reveal good agreement between the two repeated measurements. All points lie between or near the 95% confident interval lines (dotted line).

Conclusion
In this paper, sensitive, selective and robust method for quantification of MPA and MPAG metabolites in OF, plasma, and plasma ultra-filtrate is presented. Simple sample preparation and extraction protocol was developed and used to provide minimum sample dilution and appropriate samples cleanliness, excellent recovery and minimum sample components interference.

Patients Samples
After the physical examination by the physician, patients were asked to sign the informed consent. In the first study, patients were asked to give about 4mL venous blood samples, collected ethylenediaminetetraacetic acid (EDTA), and matching OF samples collected sporadically at certain time points, including, pre-dose (time 0 = C 0 ). In the second study, C 0 blood samples were collected with 3 matching OF samples collected at resting, 5 min after mouth rinsing using bottled water, and immediately after giving a saliva stimulant (commercial sour candy). Following, the patients were given vouchers for free breakfast and asked to report back at the study location shortly before 2 hours after dose (C 2 ) sampling time when blood a sample and

Clinical studies
The demographic information of studies participants is showed in Table 5.1. In total 267 samples were collected. Transferrin level higher than the recommended limit (>1mg/dL), was seen in 81 OF samples, therefore, excluded from further analysis. In the first study, intensive sampling was used to obtain blood and OF samples at rest with a total of 144 samples included in the statistical analysis. In the second study, blood and OF samples were collected at C 0 and C 2, a total of 142 samples were included. All included samples had MPA concentrations higher than the lower limit of quantification (LLOQ) Twenty-two samples had MPAG concentration lower LLOQ, but higher than the lower limit of detection (LLOD).

hours profile study
Summary statistics of MPA and MPAG concentrations in all matrices is showed in  Figure   5.3. The mean TRN concentration had an elevated level in pre-dose and started to decline and level out after one hour after the dose. In the other hand, pH level showed a random pattern.

hours profile study
In the second study, the aim was to investigate the effect of different sampling conditions on the quality of OF samples obtained before (C 0 ) and two hours (C 2 ) after taking morning medications. The samples were collected either at rest, after mouth rinsing and after giving OF stimulants. In addition, the effect of salivary blood contamination on quality and amount of the MPA and MPAG was studied. Shapiro-Wilk test revealed the abnormal distribution of salivary pH; TRN, MPA, and MPAG levels. Therefore, nonparametric tests were used.
Effect of blood salivary contamination on endogenous substances has been studied [22]. According to the authors, high TRN level was associated with higher dehydroepiandrosterone but had a mitigated effect on the salivary level of compounds studied, cortisol and testosterone. For MPA, high concentrations in OF samples collected at C 0 combined with elevated TRN level have been reported [7]. Similar finding is seen in this study, where significantly higher TRN levels in C 0 resting and rinsed samples (Figures 5.4.A and 5.4.B, respectively.) compared with C 2 resting and rinsed samples. No significant difference between stimulated OF samples collected at both time points (Figure 5.4.C).

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In addition, significant differences in TRN concentration in resting and rinsed samples compared with stimulated samples only seen in C 0 (Figure 5.5.C). However, the concentration of MPA was not significantly different between resting, rinsed, and stimulated OF samples (Figure 5.5.A), which may suggest limited/ no effect of TRN level on MPA salivary concentration. No difference in TRN level is seen in OF samples collected at C 2 (Figure 5.5.D). This may indicate the abundance of TRN in fasting samples, that even mouth rising was not enough to reduce salivary blood contamination.

Conclusion
In samples obtained from stable renal transplant recipients good correlation between AUC0-12 of MPA in OF samples and unbound and total MPA. In contrast, a weak association between MPAG concentrations in oral fluids with total and unbound plasma fraction. Limited effect of TRN level in OF on MPA concentration.

Introduction
Ghrelin is a 28-amino acid peptide primarily produced by the endocrine X/A-like cells of the fundus mucosa of the stomach and acts as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a). GHS-R1a is a G-protein coupled receptor that induces growth hormone (GH) release from the pituitary [1]. Ghrelin activates hypothalamic orexigenic neurons and inhibits anorectic neurons to induce hunger [2,3]. In humans, intravenous (IV) acetylated ghrelin administration increases appetite and food intake [4,5]. Moreover, ghrelin infusion can suppress glucose-dependent insulin secretion in rodents and humans resulting in insulin resistance [2,6].
Therefore, it is conceivable to believe that pharmacological modulation of ghrelin may be beneficial in regulating appetite and body weight or in treating type 2 diabetes mellitus.
Consistent with converging evidence illustrating that alcohol and food-seeking behaviors share common neural pathways [7,8], ghrelin signaling has been proposed as a potential novel pharmacological target for the treatment of alcoholism [9]. In mice, central ghrelin administration to reward nodes of the brain increased alcohol intake while central or peripheral administration of ghrelin receptor antagonists suppressed alcohol intake [10]. Furthermore, clinical studies from our team have shown that plasma concentrations of ghrelin were different in abstinent compared to active drinking alcohol-dependent individuals and correlated with alcohol craving [11]. Additionally, in a human laboratory setting, intravenous administration of 3 µg/kg ghrelin to alcohol-dependent, heavy-drinking individuals resulted in a significant acute increase in cue-induced alcohol craving [12]. Furthermore, there was min until the end of each run to waste. The elution time for both analyte and IS was 0.83 min.

Preparation of standards, quality controls, and IS solutions
Sub-stock and working stock solutions of PF-5190457 and IS were prepared using 50% acetonitrile (ACN) and were stored at 4 °C. Standards and quality controls (QCs) samples were prepared by spiking rat or human plasma or rat brain homogenate to achieve desired PF-5190457 concentrations while keeping the organic solvent ≤ 5%  Tables 1 and 2.

A. Rat brain samples
Brain segments from each rat were weighed individually and homogenized manually on ice using a glass tissue homogenizer with four-fold volume of de-ionized water (w:v) until a homogenous mixture was formed. One part brain homogenate of control blank, standards, QCs, and samples was extracted with two parts of 5 µg/L WIS in 1.5 mL Eppendorf tubes. Double blank samples were extracted with 100% ACN. After vortex mixing for 10 seconds, samples were centrifuged at 5000 xg for 5 min and 10 µL of supernatant was injected onto LC-MS/MS.

B. Rat and human plasma samples
One part of rat or human plasma as control blank, standards, QCs, and plasma samples was mixed with four parts of 10 ng/mL WIS in a 1.5 mL microfuge tube. Double blank samples were extracted with 100% ACN. After vortex mixing for 10 seconds, samples were centrifuged at 5000 xg for 5 min and 5µL of the supernatant was injected onto LC-MS/MS.

Standards and QCs
The method was validated in accordance with the current version of the Food and Drug Administration (FDA) guidance for industry bioanalytical method validation [14]. Calibration curves were constructed by plotting analyte/IS peak area ratio against the nominal concentration of analytes and fitted using a (1/x) weighting method.
Accuracy and precision of the assay were determined using three different batches of brain or plasma that were spiked with working stock solutions to achieve standards and QCs concentrations (6 replicate) and extracted as described in the sample extraction section.

Sensitivity and selectivity
Lower limit of quantification (LLOQ) was determined by concentrations that had % bias ≤ ± 20%, coefficient of variation (CV) ≤ ±20% and signal to noise ratio (S/N) ≤10. Acceptance criteria for QCs (LQC, MQC and HQC) was %bias ≤ ±15%and CV ≤ ±15%. Selectivity assessed by inspecting the presence of noise or peaks at analyte and IS elution time on chromatograms represented blank brain or plasma samples (from 6 subjects).

Stability
Stability of PF-5190457 was investigated by quantifying QC1 and QC3 concentrations in three replicates. Freeze and thaw (three freeze and thaw cycles), bench-top, and short-term stability for up to one month were investigated. Auto-sampler stability was assessed, by re-injecting one of the validation batches kept in the auto-sampler for over 72 hours.

Matrix effect and recovery
Possible interference of matrix effect (ME) in brain and plasma samples was inspected visually through two ways. First, possible interference of matrices components was visually inspected on chromatograms generated using post-column infusion [15]. The test was performed by continuously infusing, after the column via a Tee connection, 98% ACN solution (represents the composition of mobile phase at elution time) containing PF-5190457 and IS at highest standards concentrations at a flow rate of 10 µL/min. Simultaneously, extracted blank brain samples, plasma samples, and neat solution (%50 ACN) were injected using the pre-established LC method.
Chromatograms obtained from injecting blank brain or plasma samples were compared with a chromatogram that represented neat solution chromatograms for any signs of suppression and/or enhancement at analyte and IS elution region. Second, possible co-elution of analytes and IS with PL was also checked [16,17]. By including MRM transitions of abundant phospholipids (PL) in MS method, we were able to visually locate PL elution region at early stages of method development. Co-elution was avoided by manipulating liquid chromatography conditions and mobile phase gradients.
To determine recovery, two sets of QCs (form six subjects) were prepared. The first set of QCs was prepared in either brain or plasma and was extracted as prescribed in the samples extraction section (pre-extracted matrices QCs). The second set was prepared by spiking extracted blank matrices with standard working solutions to achieve the same final concentration as the concentration in the first set. The percentage ratio of mean peak areas of pre-extracted samples to mean post-extracted spiked samples was used to calculate recovery.

Sensitivity and selectivity
Brain concentration of analyte was expected to be very low compared to plasma. Therefore, mass spectrometry and chromatographic conditions were optimized using extracted brain samples to improve lower limit of quantification. Adequate sensitivity and selectivity were obtained using Acquity UPLC BEH C18 column. The final UPLC and mass spectrometry parameters were appropriate to set LLOQs at 0.75 and 1 µg/L for brain and plasma, respectively (Figure 4). Chromatograms obtained from pooled blank samples from six subjects and blank neat solutions (50% ACN) were visually inspected and compared for any peaks or noises at elution regions. No sign of interference was noticed. No carryover was detected when double blank samples were injected following the highest calibration concentration.
Curve fitting of the standard curve was comprised of 1/x weighted least squares linear regression. The average correlation coefficient (r 2 ) of the three validation batches was 0.999. The inter-run % bias and coefficient of variation (CV) were in the recommended limit of ±20 for LLOQ and ±15 for QCs (Table 2).

Stability
Bench top, freeze and thaw, auto-sampler, and short-term storage at -80 C o for up to four weeks were studied (Table 3). No stability problems were noticed and analytes were stable in extracted matrices for up to 72hrs.

Recovery and matrix effect
Samples processing and extraction procedures showed excellent recovery. The recovery ranged from 102-118% with CV less than 6% for all matrices (Table 3).
Endogenous components in biological fluids may interfere and compete for ionization with the analytes of interest [15]. The ME could be either ionization suppression or enhancement, both of which can potentially compromise the integrity of the data [16].
A post-column infusion technique was utilized to examine possible interference of components present in matrices of interest. Figure 5 shows a representative composite of PF-5190457 and IS traces obtained from post-column infusion at a concentration of 1 µg/mL overlaid on chromatograms obtained from injecting samples. An area of ionization suppression was seen around 0.25 minute in chromatogram from all matrices; slight ionization enhancement was also seen around 0.5 minute in all matrices ( Figure 5). There was no sign of ionization suppression or enhancement at the retention time of analyte or IS.
The ME was investigated visually first by detecting elution regions of PL components of rat brain, rat plasma and human plasma. MRM of transitions of most common PLs [16,17] were added to the mass spectrometry method. Mass transitions of PLs include m/z, 496 → 184, 520 → 184, 522 → 184, 524 → 184, 758 → 184, 782 → 184. As shown in Figure 6, the investigated PLs eluted far enough after analytes of interest in rat brain (A), rat plasma (B) and human plasma (C). It must be noted that PLs's that have m/z of 524 are more abundant in the brain when compared to rat and human plasma. In contrast, PLs's with m/z of 522 seem to be more abundant in rat and human plasma than in rat brain. Since the dilution factors (15 and 5 times for brain and plasma, respectively) and final water proportion in each final matrix extract was different, direct quantitative comparison was not possible.

Assay application
The assay was successfully utilized to measure compound concentrations in rat brains and plasma after administration of PF-5190457 as well as preliminary pharmacokinetic studies in human plasma conducted in the context of phase 1b study.
Appropriate approvals were granted by the appropriate NIH Institutional Animal Care and Use Committee (IACUC) and the Institutional Review Board (IRB). Figure 7 depicts a concentration-time profile of PF-5190457 in a representative human subject 181 at steady-state after administration of 50 and 100 mg oral dose of PF-5190457.

Conclusion
This is the first reported analytical method for quantification of PF-5190457 in rat brain, rat plasma and human plasma. This LC-MS/MS method was developed and validated in accordance with the current FDA guideline and showed high sensitivity, selectivity and robustness. Simple extraction processes with excellent recovery and sufficient sample cleanness was used. The method allowed us to examine the presence and describe relative components and elution behaviors of the investigated PLs species. The assays were successfully applied for quantification of PF-5190457 in both pre-clinical and clinical studies.  Results of stability studies