Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development and validation of a liquid-chromatography tandem mass spectrometry method to determine in vitro and in vivo histamine release Krishna C. Chimalakonda a , Eric Pang a,1 , James L. Weaver a , Kristina E. Howard a , Vikram Patel a , Michael T. Boyne II b,∗ a Center for Drug Evaluation and Research, Office of Clinical Pharmacology, Division of Applied Regulatory Science, United States Food and Drug Administration, Silver Spring, MD, USA b Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis, United States Food and Drug Administration, Silver Spring, MD, USA
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 12 October 2014 Accepted 14 October 2014 Available online 23 October 2014 Keywords: Histamine LC–MS/MS HILIC Mast cell degranulation Anaphylactic reactions
a b s t r a c t Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography–tandem mass spectrometric (LC–MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC–MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release. The LC–MS/MS procedure incorporating deuterium labeled internal standards provides rapid resolution of histamine with excellent sensitivity, precision, and accuracy. Histamine eluted at 1.5 min and was well separated from endogenous plasma peaks. The total run time of the assay was 8.0 min. A linear (r2 ≥ 0.99) instrument response over the entire concentration range of 1.0–1000 ng/mL was observed. Excellent accuracy (error ± 3.4%) and precision (CV ± 10%) of the assay was demonstrated, with the lower limit of quantitation (LLOQ) at 15.6 ng/mL. The validated LC–MS/MS assay was applied to determine histamine release in both in vitro and in vivo models. Peritoneal mast cells treated with prototypical degranulating agents (Compound 48/80 and Teicoplanin) showed that the two chemicals caused approximately 40% histamine release. In rats, using this assay, basal histamine plasma levels were typically under 100 ng/mL. Treatment with an agent suspected of causing anaphylactic type reactions resulted in plasma histamine levels to increase above 3000 ng/mL. The LC–MS/MS assay presented in this study can be applied to further characterize the physiological and pathophysiological role of histamine release in complex in vitro and in vivo models. Importantly, the LC–MS/MS assay may be useful in assessing active pharmaceutical ingredient-mediated degranulation and anaphylaxis as part of either a pre-market or a post-market assessment of drug products. © 2014 Published by Elsevier B.V.
1. Introduction
Abbreviations: LC–MS/MS, liquid-chromatography tandem mass spectrometry; HILIC, hydrophilic interaction chromatography; LLOQ, lower limit of quantitation. ∗ Corresponding author at: 10903 New Hampshire Avenue, Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis, United States Food and Drug Administration, Silver Spring, MD 20993, USA. Tel.: +1 301 796 0113; fax: +1 301 796 9859. E-mail address: [email protected] (M.T. Boyne II). 1 Current address: Center for Drug Evaluation and Research, Office of Pharmaceutical Science, Office of Generic Drugs, Division of Chemistry II, United States Food and Drug Administration, Rockville, MD, USA. http://dx.doi.org/10.1016/j.jpba.2014.10.016 0731-7085/© 2014 Published by Elsevier B.V.
Histamine is a low molecular weight amine (molecular weight: 111.15 Da; Fig. 1) that plays several critical physiological roles mediating the immune response, regulating blood vessel vasodilation, and controlling intestinal smooth muscle motility [1–3]. The most well-known role of histamine is mediating part of the inflammatory response to various allergic reactions [1,3]. Classic hypersensitivity (anaphylaxis) is mediated by release of histamine and other mediators in response to antigen cross-linking of immunoglobulin E (IgE) bound to Fc␥RI receptor on several cell types including mast cells, basophils, and neutrophils. In addition, there are alternate activation pathways not involving IgE that are caused by smaller basic molecules such as Compound 48/80 and
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
495
2. Materials and methods 2.1. Reagents and chemicals
Fig. 1. Chemical structure of histamine.
Teicoplanins that directly activate mast cells and basophils [4]. Activation of these cell types by either pathway causes the process of degranulation that results in the release of mediators including histamine and an array of inflammatory cytokines [1,3]. Clinically, histamine affects a wide range of cells and tissues with its effects mediated through four receptors [5]. The symptoms range from mild itching up to the fatal cardiovascular collapse seen in anaphylaxis. For certain drugs, like vancomycin, histamine driven adverse events are known and well described in the literature [6,7]. Previously, these adverse events were typically discovered in patients. Therefore, measurement of histamine can be important in both in vitro assays, such as mast cell activation, and in vivo samples where an anaphylactic type response is suspected. Historically, radioimmunoassay [8] (RIA), -hexosaminidase assay [9], and enzyme immunoassay [10] (EIA) were commonly used as analytical techniques for measuring histamine in rodent plasma/serum and tissues. These assays had numerous limitations, chief among them is the interference of endogenous molecules and other drugs. As histamine has no significant chromophore, commonly reported analytical methods rely on pre- or postcolumn derivatization followed by LC and fluorescence or UV detection [11]. Chromatographic separation techniques, such as gas chromatography [12], capillary electrophoresis [13], and capillary electro-chromatography [14] have also been reported. Various analytical methods were developed to quantify histamine levels in food products like fish [15], cheese [16], meat [17], and alcoholic beverages [18]. For a detailed review of the analytical methods available to determine histamine levels in these matrices, the authors would like to direct the readers to a review published by Onal et al. [19]. Reversed-phase high performance liquid chromatography (RPHPLC) analysis of histamine has been a challenge because of the high polarity of histamine, which leads to poor retention under typical reversed-phase conditions [11]. The use of ion-paring agents, such as alkane-sulphonic acid derivatives can improve the retention of small, polar bases like histamine [11], but strong ionparing additives like hexane sulphonic acid are not compatible with LC–MS/MS analysis because of their lack of volatility [11]. Hydrophilic interaction chromatography (HILIC) offers a solution for the poor retention of histamine. Under HILIC conditions, polar analytes are more strongly retained than hydrophobic analytes, thus overcoming the primary problem with the reversed-phase analysis of histamine [20,21]. Because HILIC methods use high concentration of acetonitrile as the mobile phase, it is possible to directly inject strong solvents onto the column after appropriate sample preparation without introducing peak distortion from solvent effects [20,21]. Using a HILIC based approach, Bourgogne et al. were able to detect derivatized histamine and its major metabolite from brain dialysates [22] The primary objective of the present study was to develop and validate a rapid and sensitive HILIC–MS/MS method to quantify chemically induced-histamine release in in vitro and in vivo models without the derivatization and dialysis step. In vitro release of histamine was induced by treating isolated rat peritoneal mast cells with Compound 48/80 or Teicoplanin, chemicals widely known to directly degranulate mast cells and cause histamine release [23,24]. This LC–MS/MS method was also applied to determine the plasma concentration of histamine in rats treated with an investigational chemical to demonstrate the utility of this assay for in vivo studies.
Optima grade acetonitrile and formic acid (99% pure) were purchased from Fisher Scientific (Pittsburgh, PA). Histamine hydrochloride analytical standard and deuterium-labeled internal standard (Histamine-d4 ) were purchased from Sigma Chemical Co (St. Louis, MO) and Cambridge Isotope Laboratories (Andover, MA), respectively. Compound 48/80 and Teicoplanins were purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals used in the study were of the highest reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Blank rat plasma (anticoagulant: K3 EDTA) from Sprague-Dawley rats for preparing histamine calibration standards was purchased from Innovative Research (Novi, MI).
2.2. Equipment Sample analysis from 10 L injections was performed using a Thermo Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Waltham, MA) interfaced with a Thermo 1250 Accela quaternary liquid chromatography system (Waltham, MA). Peak detection and quantification were performed using Qual Browser (Thermo Xcalibur 2.2 SP1.48). Data analysis was performed using Microsoft Excel.
2.3. Mast cell degranulation assay The mast cell degranulation assay was adapted from Shanahan et al., 1985 [25]. In brief, rat peritoneal mast cells (MC) were isolated by Percoll gradient centrifugation and re-suspended at 500,000 cells/mL in Hank’s Balanced Salt Solution (HBSS) with 1% heat-inactivated horse serum (HS, Life Technologies Corp., Carlsbad, CA) and incubated for 30 min at 37 ◦ C, 5% CO2 . Purity as assessed by toluidine blue staining was greater than 95%. Blank, control, (10 g/mL of Compound 48/80 or 500 g/mL of Teicoplanin) or an investigational drug (15 mg/mL) were diluted to a final volume of 90 L in HBSS/HS in a 96 well plate. A total of 5000 MC (10 L) was added to each well and incubated for 30 min at 37 ◦ C, 5% CO2 to allow full degranulation. The cultures were centrifuged for 5 min at 120 × g at room temperature, and 50 L of supernatant from each well was transferred to a new sample well. Five microliters of lysing reagent (5% Octyl -glucoside) was subsequently added to all cell wells. The plates containing cell lysate and supernatants were sealed and stored at −30 ◦ C until analysis. The % total histamine release = (([B]*2)/([A] − [B] + [B]*2))*100 where [A] is the histamine concentration measured in the well containing the cell lysate + ½ of the supernatant, and [B] is the histamine concentration measured in the well with ½ of the supernatant.
2.4. In vivo rat study Female Sprague-Dawley rats (10–12 weeks old) were purchased from Taconic Laboratories (Hudson, NY). Rats were fed Certified Purina Chow #5002 (Ralston Purina Co., St. Louis, MO) and water ad libitum. Whole blood from rats treated with an investigational drug was collected via in-dwelling cannulas using procedures approved by the Institutional Animal Care and Use Committee, Center for Drug Evaluation and Research, FDA, and carried out in an AAALACaccredited facility. Plasma from EDTA anti-coagulated blood was collected via centrifugation and stored at −30 ◦ C until analysis.
496
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
Table 1 MS/MS experimental conditions for selected reaction monitoring (SRM) for the quantitation of Histamine. Analyte
Q1 (m/z)
Q3 (m/z)
Collision energy (V)
Resolution
AGC target
Ion isolation (m/z)
Histamine Histamine-d4
112 116
94 98
80 80
70,000 70,000
5 × 106 5 × 106
0.4 0.4
2.5. Liquid chromatography/mass spectrometry conditions Histamine was chromatographically separated under isocratic conditions using a Kinetex HILIC column (2.6 M, 100 A, 50 mm × 2.1 mm, Phenomenex, CA) at 700 L/min flow rate and heated to 60 ◦ C. Mobile phases consisted of solvent A (0.1% formic acid in acetonitrile) and solvent B (20 mM ammonium formate with 0.2% formic acid). A step gradient comprised of two isocratic
portions was used: 25% solvent A for 2 min, then 70% solvent A for 1.5 min. Finally the column was re-equilibrated to initial conditions. The total run time was 5 min. Histamine and Histamine-d4 were detected on a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer equipped with HESI ion source at 70k resolution at 200 m/z. The AGC target was set at 5 × 106 and the maximum injection time at 50 ms. The ion isolation was 0.4 m/z with collision energy of 80 eV. The fragment ions
Fig. 2. LC–MS/MS chromatograms of a plasma sample taken from a rat dosed with vehicle (blank; panel A, containing endogenous histamine), lowest spiked plasma QC (15.6 ng/mL; panel B), and after the administration of a single 15 mg/mL dose of an investigational drug (panel C). Please note differences in the relative abundance-scale on the Y-axis.
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
are monitored for histamine (m/z = 112.087 > 95.061 Da) and for deuterium-labeled histamine (m/z = 116.112 > 99.086 Da). A summary of the mass spectrometric parameters is presented in Table 1. 2.6. Preparation of analytical standards and quality controls Analytical standards were prepared using a calibration stock solution containing histamine (100 g/mL) in water that was stored at −30 ◦ C until use. Daily calibration standards (blank (0), 0.98, 3.9, 15.6, 62.5, 250, and 1000 ng/mL) were prepared in HBSS or blank rat plasma by first preparing an intermediate working solution in water (5 g/mL), followed by serial dilution with blank HBSS or blank rat plasma to yield final calibration standards ranging from 1.0 to 1000 ng/mL. Quality control (QC) samples were prepared independent of standards by spiking varying levels of histamine (15.6, 250, and 1000 ng/mL; final concentrations) in blank HBSS or blank rat plasma. 2.7. Sample preparation of analytical standards, quality controls, mast cell and rat plasma samples from the in vitro and in vivo study 50 L of histamine calibration standard in HBSS or 50 L mast cell supernatant/pellet or 100 L of histamine calibration standard in blank rat plasma or plasma from rats treated with an investigational drug were mixed with 3 volumes of acetonitrile, containing 0.1% formic acid and 100 ng/mL deuterium labeled internal standard. The standards and samples were vortexed for 10 s and the mixture was spun down at 3000 × g at 4 ◦ C for 10 min. Subsequently, the top 90% of the supernatant was transferred into sample vials, and 10 L was injected onto the column and analyzed as described above. The inter-run accuracy (% error) and precision (% CV) of the assays were determined from the analysis of quality control samples (n = 5) based on reported US FDA guidelines [26,27]. Accuracy and precision measurements for quality controls were assessed by interday (nonconsecutive), replicate analysis (N = 5) of rat plasma histamine quality control (QC) samples. Accuracy was calculated as the absolute percent relative error for each of the expected QC concentrations using the following equation: Accuracy =
[nominal concentration − mean calculated concentration] × 100 [nominal concentration]
Analytical precision was calculated as the coefficient of variation (%CV) for replicate measurements at the three QC concentrations (15.6, 250, and 1000 ng/mL).
497
Table 2 Interday accuracy and precision for quantitation of histamine in rat plasma (n = 5). Added Conc. (ng/mL)
Mean Calculated Conc. (ng/mL)
CV (%)
Error (%)
15.6 250 1000
15.1 245 1018
9.4 5.7 2.9
−3.4 −1.9 1.8
validation, auto-sampler carryover was not detected while monitoring for the analytes of interest. A linear instrument response (IR = Peak AreaAnalyte /Peak AreaIS ) over the calibration range (1.0–1000 ng/mL) was observed in all experiments when a linear least-squares regression with linear weighting was used to calculate a line of best fit. Validation results for the histamine assay in rat plasma are presented in Table 2. Accuracy (% error) and precision (CV) of the assay was determined to be within ±3.4% and ±10% for all the QC samples, respectively. On the basis of the data presented in Table 2, the lower limit of quantitation of histamine is 15.6 ng/mL where LLOQ = 10/S where S = the slope of the calibration curve and is the residual standard deviation of a regression line. 3.2. Selectivity, reproducibility, and stability Selectivity of the histamine assay was determined through elution time and selective reaction monitoring (SRM). Hydrophilic interaction chromatography (HILIC) was used to separate and detect histamine. The LLOQ calculated based on both fragments yield similar results. The day-to-day variability in the assay was shown to be less than 15% for the concentrations tested between 1.0–1000 ng/mL. Additionally, bench top (room temperature 25 ◦ C for 1 h) and freeze–thaw (2 cycles; −30 ◦ C) stability of histamine at two concentrations (15.6 and 1000 ng/mL) were determined and no significant loss (>15%) in histamine concentration was observed (data not shown). 3.3. Assessing in vitro and in vivo Histamine Release The validated histamine HILIC–MS/MS assay was successfully applied to determine in vitro histamine release in rat peritoneal mast cells treated with Compound 48/80 and Teicoplanin, which directly degranulate mast cells and cause histamine release. Fig. 3 shows the in vitro histamine release after isolated rat mast cells were treated with 10 g/mL of Compound 48/80 and 500 g/mL of
3. Results 3.1. Histamine assay characterization LC–MS/MS chromatograms of a plasma sample taken from a rat dosed with vehicle (blank; containing endogenous histamine), lowest spiked plasma QC (15.6 ng/mL) and after the administration of a single 15 mg/mL dose of an investigational drug are shown in Fig. 2A–C. Histamine eluted at 1.5 min, and was well separated from the endogenous peaks in plasma, with a maximum ±0.1 minute variation between runs (Figure 2). The width of the histamine elution peak was ∼0.3 min. The LC–MS/MS procedure provided baseline resolution of histamine and retention times that were consistent between all standards, QCs, and samples and retention time did not shift (not greater than 0.1 min) upon subsequent injections. Auto-sampler carryover was assessed by injecting blank plasma samples that did not contain any standard material and solvent blanks directly after the analysis of high calibration standard and quality control samples (1000 ng/mL). Throughout method
Fig. 3. Compound 48/80 and teicoplanin induced-histamine release in vitro.
498
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
4. Discussion
Fig. 4. Viability of rat peritoneal mast cells incubated for 30 min in test solutions. Squares – HBSS, circles – test buffer.
Teicoplanin, respectively. Both compounds caused approximately 40% total histamine release in rat mast cells, which was significantly greater in comparison to saline controls (
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Development and validation of a liquid-chromatography tandem mass spectrometry method to determine in vitro and in vivo histamine release Krishna C. Chimalakonda a , Eric Pang a,1 , James L. Weaver a , Kristina E. Howard a , Vikram Patel a , Michael T. Boyne II b,∗ a Center for Drug Evaluation and Research, Office of Clinical Pharmacology, Division of Applied Regulatory Science, United States Food and Drug Administration, Silver Spring, MD, USA b Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis, United States Food and Drug Administration, Silver Spring, MD, USA
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 12 October 2014 Accepted 14 October 2014 Available online 23 October 2014 Keywords: Histamine LC–MS/MS HILIC Mast cell degranulation Anaphylactic reactions
a b s t r a c t Histamine is an important biogenic amine involved in regulating numerous physiological and pathophysiological processes in humans and animals. To date, there have been very few studies focused on developing and validating sensitive liquid-chromatography–tandem mass spectrometric (LC–MS/MS) assays capable of quantitative trace level histamine analysis in biological matrices. In the present study, a rapid and sensitive LC–MS/MS assay, amenable to high throughput analysis was developed and validated to characterize in vitro and in vivo histamine release. The LC–MS/MS procedure incorporating deuterium labeled internal standards provides rapid resolution of histamine with excellent sensitivity, precision, and accuracy. Histamine eluted at 1.5 min and was well separated from endogenous plasma peaks. The total run time of the assay was 8.0 min. A linear (r2 ≥ 0.99) instrument response over the entire concentration range of 1.0–1000 ng/mL was observed. Excellent accuracy (error ± 3.4%) and precision (CV ± 10%) of the assay was demonstrated, with the lower limit of quantitation (LLOQ) at 15.6 ng/mL. The validated LC–MS/MS assay was applied to determine histamine release in both in vitro and in vivo models. Peritoneal mast cells treated with prototypical degranulating agents (Compound 48/80 and Teicoplanin) showed that the two chemicals caused approximately 40% histamine release. In rats, using this assay, basal histamine plasma levels were typically under 100 ng/mL. Treatment with an agent suspected of causing anaphylactic type reactions resulted in plasma histamine levels to increase above 3000 ng/mL. The LC–MS/MS assay presented in this study can be applied to further characterize the physiological and pathophysiological role of histamine release in complex in vitro and in vivo models. Importantly, the LC–MS/MS assay may be useful in assessing active pharmaceutical ingredient-mediated degranulation and anaphylaxis as part of either a pre-market or a post-market assessment of drug products. © 2014 Published by Elsevier B.V.
1. Introduction
Abbreviations: LC–MS/MS, liquid-chromatography tandem mass spectrometry; HILIC, hydrophilic interaction chromatography; LLOQ, lower limit of quantitation. ∗ Corresponding author at: 10903 New Hampshire Avenue, Center for Drug Evaluation and Research, Division of Pharmaceutical Analysis, United States Food and Drug Administration, Silver Spring, MD 20993, USA. Tel.: +1 301 796 0113; fax: +1 301 796 9859. E-mail address: [email protected] (M.T. Boyne II). 1 Current address: Center for Drug Evaluation and Research, Office of Pharmaceutical Science, Office of Generic Drugs, Division of Chemistry II, United States Food and Drug Administration, Rockville, MD, USA. http://dx.doi.org/10.1016/j.jpba.2014.10.016 0731-7085/© 2014 Published by Elsevier B.V.
Histamine is a low molecular weight amine (molecular weight: 111.15 Da; Fig. 1) that plays several critical physiological roles mediating the immune response, regulating blood vessel vasodilation, and controlling intestinal smooth muscle motility [1–3]. The most well-known role of histamine is mediating part of the inflammatory response to various allergic reactions [1,3]. Classic hypersensitivity (anaphylaxis) is mediated by release of histamine and other mediators in response to antigen cross-linking of immunoglobulin E (IgE) bound to Fc␥RI receptor on several cell types including mast cells, basophils, and neutrophils. In addition, there are alternate activation pathways not involving IgE that are caused by smaller basic molecules such as Compound 48/80 and
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
495
2. Materials and methods 2.1. Reagents and chemicals
Fig. 1. Chemical structure of histamine.
Teicoplanins that directly activate mast cells and basophils [4]. Activation of these cell types by either pathway causes the process of degranulation that results in the release of mediators including histamine and an array of inflammatory cytokines [1,3]. Clinically, histamine affects a wide range of cells and tissues with its effects mediated through four receptors [5]. The symptoms range from mild itching up to the fatal cardiovascular collapse seen in anaphylaxis. For certain drugs, like vancomycin, histamine driven adverse events are known and well described in the literature [6,7]. Previously, these adverse events were typically discovered in patients. Therefore, measurement of histamine can be important in both in vitro assays, such as mast cell activation, and in vivo samples where an anaphylactic type response is suspected. Historically, radioimmunoassay [8] (RIA), -hexosaminidase assay [9], and enzyme immunoassay [10] (EIA) were commonly used as analytical techniques for measuring histamine in rodent plasma/serum and tissues. These assays had numerous limitations, chief among them is the interference of endogenous molecules and other drugs. As histamine has no significant chromophore, commonly reported analytical methods rely on pre- or postcolumn derivatization followed by LC and fluorescence or UV detection [11]. Chromatographic separation techniques, such as gas chromatography [12], capillary electrophoresis [13], and capillary electro-chromatography [14] have also been reported. Various analytical methods were developed to quantify histamine levels in food products like fish [15], cheese [16], meat [17], and alcoholic beverages [18]. For a detailed review of the analytical methods available to determine histamine levels in these matrices, the authors would like to direct the readers to a review published by Onal et al. [19]. Reversed-phase high performance liquid chromatography (RPHPLC) analysis of histamine has been a challenge because of the high polarity of histamine, which leads to poor retention under typical reversed-phase conditions [11]. The use of ion-paring agents, such as alkane-sulphonic acid derivatives can improve the retention of small, polar bases like histamine [11], but strong ionparing additives like hexane sulphonic acid are not compatible with LC–MS/MS analysis because of their lack of volatility [11]. Hydrophilic interaction chromatography (HILIC) offers a solution for the poor retention of histamine. Under HILIC conditions, polar analytes are more strongly retained than hydrophobic analytes, thus overcoming the primary problem with the reversed-phase analysis of histamine [20,21]. Because HILIC methods use high concentration of acetonitrile as the mobile phase, it is possible to directly inject strong solvents onto the column after appropriate sample preparation without introducing peak distortion from solvent effects [20,21]. Using a HILIC based approach, Bourgogne et al. were able to detect derivatized histamine and its major metabolite from brain dialysates [22] The primary objective of the present study was to develop and validate a rapid and sensitive HILIC–MS/MS method to quantify chemically induced-histamine release in in vitro and in vivo models without the derivatization and dialysis step. In vitro release of histamine was induced by treating isolated rat peritoneal mast cells with Compound 48/80 or Teicoplanin, chemicals widely known to directly degranulate mast cells and cause histamine release [23,24]. This LC–MS/MS method was also applied to determine the plasma concentration of histamine in rats treated with an investigational chemical to demonstrate the utility of this assay for in vivo studies.
Optima grade acetonitrile and formic acid (99% pure) were purchased from Fisher Scientific (Pittsburgh, PA). Histamine hydrochloride analytical standard and deuterium-labeled internal standard (Histamine-d4 ) were purchased from Sigma Chemical Co (St. Louis, MO) and Cambridge Isotope Laboratories (Andover, MA), respectively. Compound 48/80 and Teicoplanins were purchased from Sigma Chemical Co (St. Louis, MO). All other chemicals used in the study were of the highest reagent grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Blank rat plasma (anticoagulant: K3 EDTA) from Sprague-Dawley rats for preparing histamine calibration standards was purchased from Innovative Research (Novi, MI).
2.2. Equipment Sample analysis from 10 L injections was performed using a Thermo Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer (Waltham, MA) interfaced with a Thermo 1250 Accela quaternary liquid chromatography system (Waltham, MA). Peak detection and quantification were performed using Qual Browser (Thermo Xcalibur 2.2 SP1.48). Data analysis was performed using Microsoft Excel.
2.3. Mast cell degranulation assay The mast cell degranulation assay was adapted from Shanahan et al., 1985 [25]. In brief, rat peritoneal mast cells (MC) were isolated by Percoll gradient centrifugation and re-suspended at 500,000 cells/mL in Hank’s Balanced Salt Solution (HBSS) with 1% heat-inactivated horse serum (HS, Life Technologies Corp., Carlsbad, CA) and incubated for 30 min at 37 ◦ C, 5% CO2 . Purity as assessed by toluidine blue staining was greater than 95%. Blank, control, (10 g/mL of Compound 48/80 or 500 g/mL of Teicoplanin) or an investigational drug (15 mg/mL) were diluted to a final volume of 90 L in HBSS/HS in a 96 well plate. A total of 5000 MC (10 L) was added to each well and incubated for 30 min at 37 ◦ C, 5% CO2 to allow full degranulation. The cultures were centrifuged for 5 min at 120 × g at room temperature, and 50 L of supernatant from each well was transferred to a new sample well. Five microliters of lysing reagent (5% Octyl -glucoside) was subsequently added to all cell wells. The plates containing cell lysate and supernatants were sealed and stored at −30 ◦ C until analysis. The % total histamine release = (([B]*2)/([A] − [B] + [B]*2))*100 where [A] is the histamine concentration measured in the well containing the cell lysate + ½ of the supernatant, and [B] is the histamine concentration measured in the well with ½ of the supernatant.
2.4. In vivo rat study Female Sprague-Dawley rats (10–12 weeks old) were purchased from Taconic Laboratories (Hudson, NY). Rats were fed Certified Purina Chow #5002 (Ralston Purina Co., St. Louis, MO) and water ad libitum. Whole blood from rats treated with an investigational drug was collected via in-dwelling cannulas using procedures approved by the Institutional Animal Care and Use Committee, Center for Drug Evaluation and Research, FDA, and carried out in an AAALACaccredited facility. Plasma from EDTA anti-coagulated blood was collected via centrifugation and stored at −30 ◦ C until analysis.
496
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
Table 1 MS/MS experimental conditions for selected reaction monitoring (SRM) for the quantitation of Histamine. Analyte
Q1 (m/z)
Q3 (m/z)
Collision energy (V)
Resolution
AGC target
Ion isolation (m/z)
Histamine Histamine-d4
112 116
94 98
80 80
70,000 70,000
5 × 106 5 × 106
0.4 0.4
2.5. Liquid chromatography/mass spectrometry conditions Histamine was chromatographically separated under isocratic conditions using a Kinetex HILIC column (2.6 M, 100 A, 50 mm × 2.1 mm, Phenomenex, CA) at 700 L/min flow rate and heated to 60 ◦ C. Mobile phases consisted of solvent A (0.1% formic acid in acetonitrile) and solvent B (20 mM ammonium formate with 0.2% formic acid). A step gradient comprised of two isocratic
portions was used: 25% solvent A for 2 min, then 70% solvent A for 1.5 min. Finally the column was re-equilibrated to initial conditions. The total run time was 5 min. Histamine and Histamine-d4 were detected on a Q-Exactive Hybrid Quadrupole Orbitrap mass spectrometer equipped with HESI ion source at 70k resolution at 200 m/z. The AGC target was set at 5 × 106 and the maximum injection time at 50 ms. The ion isolation was 0.4 m/z with collision energy of 80 eV. The fragment ions
Fig. 2. LC–MS/MS chromatograms of a plasma sample taken from a rat dosed with vehicle (blank; panel A, containing endogenous histamine), lowest spiked plasma QC (15.6 ng/mL; panel B), and after the administration of a single 15 mg/mL dose of an investigational drug (panel C). Please note differences in the relative abundance-scale on the Y-axis.
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
are monitored for histamine (m/z = 112.087 > 95.061 Da) and for deuterium-labeled histamine (m/z = 116.112 > 99.086 Da). A summary of the mass spectrometric parameters is presented in Table 1. 2.6. Preparation of analytical standards and quality controls Analytical standards were prepared using a calibration stock solution containing histamine (100 g/mL) in water that was stored at −30 ◦ C until use. Daily calibration standards (blank (0), 0.98, 3.9, 15.6, 62.5, 250, and 1000 ng/mL) were prepared in HBSS or blank rat plasma by first preparing an intermediate working solution in water (5 g/mL), followed by serial dilution with blank HBSS or blank rat plasma to yield final calibration standards ranging from 1.0 to 1000 ng/mL. Quality control (QC) samples were prepared independent of standards by spiking varying levels of histamine (15.6, 250, and 1000 ng/mL; final concentrations) in blank HBSS or blank rat plasma. 2.7. Sample preparation of analytical standards, quality controls, mast cell and rat plasma samples from the in vitro and in vivo study 50 L of histamine calibration standard in HBSS or 50 L mast cell supernatant/pellet or 100 L of histamine calibration standard in blank rat plasma or plasma from rats treated with an investigational drug were mixed with 3 volumes of acetonitrile, containing 0.1% formic acid and 100 ng/mL deuterium labeled internal standard. The standards and samples were vortexed for 10 s and the mixture was spun down at 3000 × g at 4 ◦ C for 10 min. Subsequently, the top 90% of the supernatant was transferred into sample vials, and 10 L was injected onto the column and analyzed as described above. The inter-run accuracy (% error) and precision (% CV) of the assays were determined from the analysis of quality control samples (n = 5) based on reported US FDA guidelines [26,27]. Accuracy and precision measurements for quality controls were assessed by interday (nonconsecutive), replicate analysis (N = 5) of rat plasma histamine quality control (QC) samples. Accuracy was calculated as the absolute percent relative error for each of the expected QC concentrations using the following equation: Accuracy =
[nominal concentration − mean calculated concentration] × 100 [nominal concentration]
Analytical precision was calculated as the coefficient of variation (%CV) for replicate measurements at the three QC concentrations (15.6, 250, and 1000 ng/mL).
497
Table 2 Interday accuracy and precision for quantitation of histamine in rat plasma (n = 5). Added Conc. (ng/mL)
Mean Calculated Conc. (ng/mL)
CV (%)
Error (%)
15.6 250 1000
15.1 245 1018
9.4 5.7 2.9
−3.4 −1.9 1.8
validation, auto-sampler carryover was not detected while monitoring for the analytes of interest. A linear instrument response (IR = Peak AreaAnalyte /Peak AreaIS ) over the calibration range (1.0–1000 ng/mL) was observed in all experiments when a linear least-squares regression with linear weighting was used to calculate a line of best fit. Validation results for the histamine assay in rat plasma are presented in Table 2. Accuracy (% error) and precision (CV) of the assay was determined to be within ±3.4% and ±10% for all the QC samples, respectively. On the basis of the data presented in Table 2, the lower limit of quantitation of histamine is 15.6 ng/mL where LLOQ = 10/S where S = the slope of the calibration curve and is the residual standard deviation of a regression line. 3.2. Selectivity, reproducibility, and stability Selectivity of the histamine assay was determined through elution time and selective reaction monitoring (SRM). Hydrophilic interaction chromatography (HILIC) was used to separate and detect histamine. The LLOQ calculated based on both fragments yield similar results. The day-to-day variability in the assay was shown to be less than 15% for the concentrations tested between 1.0–1000 ng/mL. Additionally, bench top (room temperature 25 ◦ C for 1 h) and freeze–thaw (2 cycles; −30 ◦ C) stability of histamine at two concentrations (15.6 and 1000 ng/mL) were determined and no significant loss (>15%) in histamine concentration was observed (data not shown). 3.3. Assessing in vitro and in vivo Histamine Release The validated histamine HILIC–MS/MS assay was successfully applied to determine in vitro histamine release in rat peritoneal mast cells treated with Compound 48/80 and Teicoplanin, which directly degranulate mast cells and cause histamine release. Fig. 3 shows the in vitro histamine release after isolated rat mast cells were treated with 10 g/mL of Compound 48/80 and 500 g/mL of
3. Results 3.1. Histamine assay characterization LC–MS/MS chromatograms of a plasma sample taken from a rat dosed with vehicle (blank; containing endogenous histamine), lowest spiked plasma QC (15.6 ng/mL) and after the administration of a single 15 mg/mL dose of an investigational drug are shown in Fig. 2A–C. Histamine eluted at 1.5 min, and was well separated from the endogenous peaks in plasma, with a maximum ±0.1 minute variation between runs (Figure 2). The width of the histamine elution peak was ∼0.3 min. The LC–MS/MS procedure provided baseline resolution of histamine and retention times that were consistent between all standards, QCs, and samples and retention time did not shift (not greater than 0.1 min) upon subsequent injections. Auto-sampler carryover was assessed by injecting blank plasma samples that did not contain any standard material and solvent blanks directly after the analysis of high calibration standard and quality control samples (1000 ng/mL). Throughout method
Fig. 3. Compound 48/80 and teicoplanin induced-histamine release in vitro.
498
K.C. Chimalakonda et al. / Journal of Pharmaceutical and Biomedical Analysis 102 (2015) 494–499
4. Discussion
Fig. 4. Viability of rat peritoneal mast cells incubated for 30 min in test solutions. Squares – HBSS, circles – test buffer.
Teicoplanin, respectively. Both compounds caused approximately 40% total histamine release in rat mast cells, which was significantly greater in comparison to saline controls (
