Journal of Chromatographic Science Advance Access published June 11, 2014 Journal of Chromatographic Science 2014;1– 10 doi:10.1093/chromsci/bmu056

Article

An Optimized Method for Corticosterone Analysis in Mouse Plasma by Ultra-Performance Liquid Chromatography-Full-Scan High-Resolution Accurate Mass Spectrometry Hui Li1, Li-Ping Sheng1, Bing Wang1, Zong-Lin Yang1 and Shu-Ying Liu1,2* 1 Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, PR China, and 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China

*Author to whom correspondence should be addressed. Email: [email protected] Received 11 September 2013; revised 9 April 2014

A simple method based on liquid chromatography – full-scan highresolution accurate mass spectrometry (LC – HRMS) using a quadrupole time-of-flight (TOF) mass spectrometry was developed and optimized for corticosterone quantification in mouse plasma. Mouse plasma (100 mL) was extracted with methyl tert-butyl ether using prednisone as internal standard. Separation was performed on a short C18 column using a methanol – water gradient. Full-scan data were acquired in the TOF only mode, and extracted ion chromatograms were generated post-acquisition with the extract masses of the analytes. Enhanced sensitivity and reproducibility were acquired with optimized mass parameters. The calibration range was 8.24 – 412 ng/mL, and the limit of quantitation was 5.088 g/mL. Accuracy was between 25.9 and 8.6%. The precision of between-run (interday) and within-run (intraday) was within 5.6 and 6.9%, respectively. The LC– HRMS method was applied for plasma samples analysis from the stressed mice with and without ginseng treatment for the stress state estimation.

Introduction Recently, liquid chromatography – tandem mass spectrometry (LC – MS) has played a key role in bioanalytical laboratories for the quantification of small molecule drugs, their metabolites and other xenobiotic compounds from biological samples including plasma, blood, serum, urine and tissue (1 – 4). LC – MS with triple quadrupole mass spectrometry (MS), which has been the workhorse of bioanalytical laboratories for nearly two decades, has excellent selectivity, good precision, noteworthy accuracy and wide dynamic ranges (1). However, scientists have become aware of some of the pitfalls of LC–MS bioanalytical methods that include potential metabolite interference and ionization suppression/enhancement, also known as matrix effect. Therefore, appropriate precautions are being taken during method development and application to avoid these shortcomings. LC coupled with high-resolution accurate mass spectrometry (HRMS) has made a huge impact in a number of analytical fields. LC – HRMS differs from LC –MS in that total ion chromatograms (TICs) are acquired over a predefined m/z range with a preset mass resolution. Extracted ion chromatograms (EICs) are subsequently obtained post-data acquisition from TIC, together with the extract masses of the target analytes and a predefined mass extraction window (MEW) against that of selected reaction monitoring -based methods in LC –MS (5). Through a single platform like LC – HRMS that combines selectivity and sensitivity, both quantitative and qualitative information could be collected simultaneously from the samples.

LC–HRMS technology has been successfully used for protein modification, peptide mapping, metabolomics and biomarker discovery (6). Higher mass resolution often provides better selectivity, especially in complex sample matrix (7, 8). The ability of LC–HRMS to detect both the ions ( protonated, sodium or ammonium adducted, etc.) generated from the target analytes in addition to ions generated from other compounds in the injected sample is unbeatable (9, 10). This approach has also been successfully applied to residue analysis of pesticides and analysis of veterinary drugs in animal-based food (11). There are basically two platforms that can be used for LC – HRMS, such as those based on the orbitrap technology and those based on the time-of-flight (TOF) technology (12). Orbitrap-based mass systems generally provide better selectivity than quadrupole TOF (Q-TOF) systems; however, this comes at the expense of increasing scan time (13). Although Q-TOF MS is a powerful detection system which was commonly used for qualitative analysis of target compounds in highly complex matrixes in metabolomics, its use in quantitative analysis is rare (14). The objective of this study was to assess the feasibility of Q-TOF MS for quantitative applications. Therefore, we evaluated the performance of Q-TOF on the corticosterone (CORT) quantitation in mouse plasma. CORT is an important glucocorticoid, which is synthesized and secreted from the adrenal medulla. Stress-related activation of the hypothalamic – pituitary – adrenal axis characterized with an increase in plasma CORT levels in rodents is an important manifestation of the physiological stress response. It is, however, often needed to measure the CORT concentrations under many circumstances. Numerous LC – MS methods have been reported for CORT determination by LC – MS (15 – 17) with the lower limit of quantitation ranging from 0.03 ng to 1.00 ng/mL, respectively. Herein, we present our evaluation of the LC–HRMS method with Q-TOF MS for the determination of CORT in mouse plasma with optimized spectrometry and ionization parameters with selectivity, accuracy, precision and incurred sample analysis.

Experimental Materials and reagents Reference standard of CORT ( purity 98%) was purchased from Sigma [internal standard (IS), St. Louis, MO, USA]. Prednisone ( purity 98%) (IS) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Chemical structures of CORT and IS were given in (Figure 1). Methanol (HPLC grade), acetonitrile (HPLC grade) and formic acid (analysis grade) were

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(N2), nebulizer pressure 35 psi (N2) and Vcap 3,500 V. For the TOF experiments, the fragmentor was set at 120 V, skimmer at 65 V and OCT 1 RF Vpp at 750 V, the mass range scanned was 100 – 1,000 m/z, at the scan rate of 2 spectra/s. The resolution was set at 10,000. Data were acquired on the centroid mode. The EICs of CORT were obtained with the exact mass with an MEW of 50 ppm.

Figure 1. Chemical structures of CORT and IS.

purchased from Tedia (Fairfield, OH, USA). Distilled water was deionized and purified by a Milli-Q purification system (18.2 VM cm, Millipore, Molsheim, France). Methyl tert-butyl ether (MTBE) (analysis grade) was purchased from TCI development Co., Ltd (Shanghai, China).

Mouse treatment Male Kunming mice (18 –22 g) supplied by the Jilin University, China with approval No. SCXK (Jilin)-(2011-0003) were used in the present study. The mice were divided into three groups: normal group, stressed group and ginseng-treated stressed group with 12 mice in each group. They were kept in a temperature controlled room (258C) with a 12-h light cycle and acclimatized for 1 week prior to study. Blood was collected from retro-orbital venous plexus to centrifuge tube painted with EDTA-3K (between 11 : 00 and 12:00 am), then placed into a fresh eppendorf tube and stored at 2808C until assayed. All animal handling procedures were performed in compliance with the PR China legislation for the use and care of laboratory animals.

Methods LC –HRMS analysis An Agilent 1200 Rapid Resolution LC series consisted of a binary pump, an autosampler, a degasser, an automatic thermostatic column compartment and a computer with an Agilent ChemStation software revision B.03.01 (Agilent Technologies) were used for LC separation. Chromatographic separation was performed with a reversed-phase Aldrich C18 (50  3 mm) with a particle size of 2.7 mm (Sigma). The mobile phase consisted of water with formic acid (0.1%) and methanol gradient was filtered (0.45 mm) before use. Gradient of the mobile phase was methanol –water (30 : 70, v/v) from time t ¼ 0 then linear to (50 : 50, v/v) at t 1 min and maintained till t ¼ 5 min then linear to (100 : 0, v/v) at t ¼ 10 min. Post-run-time was set 6 min for equilibrium. Column temperature was maintained at 35 + 0.58C. The flow rate was set at 0.6 mL/min. Detection was accomplished at t ¼ 10 min. An Agilent 6520 Accurate-Mass Q-TOF mass spectrometer was used in positive ion mode coupled with atmospheric pressure chemical ionization (APCI) interface. The source conditions were set as follows: gas temperature 3508C, drying gas 4 L/min 2 Li et al.

Extraction procedures Extraction procedures were performed as follows: an aliquot of 100 mL plasma sample (or blank, or calibrators or quality control (QC) samples) was added into 1.5 mL polypropylene tubes, which contained 10 mL IS solution (618 ng/mL in methanol) and 100 mL 0.1 M phosphate followed by vortex mixed. Then, 1 mL MTBE was added for mixing for another 15 s3 again and centrifuged at 4,000 rpm for 5 min to separate organic phase. Furthermore, the supernatant was transferred into another 1.5 mL tube then evaporated to dryness and redissolved in 100 mL methanol –water (80 : 20, v/v) solution and then was centrifuged at 12,000 rpm for 10 min again. Ten microliters of the aliquots were injected onto the chromatograph for analysis.

Preparation of blank plasma Charcoal-treated mouse plasma was used to prepare standard samples so as to remove interference of endogenous CORT. Pooled plasma was stirred at room temperature with 20% charcoal for 1 h. Later, the plasma was centrifuged at 5,000 rpm for 30 min at 58C and then filtered through a 0.5-mm filter to remove carbon particles (18).

Preparation of stock solution, calibration standards and QC samples Primary CORT stock solutions for preparation of standard and QC samples were prepared as follows. A stock solution of CORT was prepared at a concentration of 2 mg/mL in methanol and was diluted stepwise with methanol to get the working standard solutions of concentrations 84.8, 164.8, 412, 824, 2,060 and 4,120 ng/mL. The primary stock solution of IS was also prepared in methanol (1 mg/mL) and diluted to obtain a working solution of 618 ng/mL. All the solutions were kept at 48C and brought to room temperature before use. Standard curve was prepared with 10 mL standard solutions added to plasma (100 mL). Calibration plots were constructed in the range of 8.48 –412 ng/mL in mouse plasma. QC samples were prepared in the same way as the calibration standards to obtain three different plasma concentrations (8.48, 82.4 and 412 ng/mL). The calibration standards and QC samples were stored at 2208C and were brought to room temperature before use.

Method validation The method was validated in accordance with current acceptance criteria for bioanalytical method validation accepted by Food and Drug Administration (19). Selectivity was evaluated by analyzing mouse plasma from charcoal-treated sources to investigate the potential interferences as well as the real sample

and the spiked real sample at the retention times of CORT and IS. The MS/MS spectra with structure assignment of CORT were also compared both in real plasma sample and CORT solution. The acceptance criterion for experiment was that there were no significant interfering peaks at the retention time of the analysis. Calibration curves were obtained by analyzing standard plasma samples at six concentrations of 8.48, 16.48, 41.2, 82.4, 206 and 412 ng/mL. The linear regression equation was y ¼ ax þ b, in which y was the extract peak area ratio of CORT to IS and x was the concentration of CORT. The limit of detection (LOD) was defined as the sample concentration of CORT resulting in a peak height of three times signal to noise (S/N). The LOQ was defined as the sample concentration of CORT resulting in a peak height of 10 times S/N. The within-run (intraday) and between-run (interday) precisions were expressed as relative coefficient variation (CV), and the accuracy was expressed as relative error (RE). Within-run assay on each day consisted of a set of calibration stands and three batched of QC samples at three concentrations (8.48, 41.2 and 412 ng/mL, n ¼ 3) against a separate calibration curve. The between-run precisions were evaluated by analysis of three batches of QC samples on 5 different days. The accuracy was evaluated as percentage error (mean of measured-mean of added)/mean of added. The accuracy and precision were required to be within +15% of the nominal concentration and ,15% CV for all three concentration levels of QC samples. The recovery was calculated by comparing the response of CORT from charcoal-treated plasma spiked with 164.8, 824 and 4,120 ng/mL of CORT solution before extraction processed as described earlier versus response of CORT of the same concentration obtained by spiking the methanol standard solution 10 mL to IS solution into organic layer after extraction then reconstituted. Each sample was analyzed for five times. The matrix effect was assessed by comparison of the mean peak areas of the analytes at three QC concentrations spiked into post-extraction blank plasma extracts originating from six different mice with the mean peak areas for neat solutions of the analytes in 80% methanol. The stability of CORT in spiked mouse plasma was investigated under various conditions with every five replicates of QC samples on three different concentrations. Short-term stability was assessed by placing samples on the bench at ambient temperature for 4 h. Long-term stability was evaluated after the samples had been stored at 2808C for 30 days. Autosampler stability was examined by placing processed QC samples in an autosampler at room temperature for 16 h, and freeze –thaw stability was determined by freezing QC samples completely at 2808C and afterwards thawing completely again at room temperature for three cycles. The samples were considered to be stable if the assay values were within the acceptance limits of accuracy (+15% RE) and precision (+15% CV).

detection parameters in order to enhance sensitivity and reproducibility for detection.

Comparison of ion sources for electrospray ionizationand APCI The main used ionization techniques for MS are electrospray ionization (ESI) and APCI. For corticosteroids detection, ESI was commonly reported one (20). At the start of method development, response signals with ESI and APCI were evaluated by comparison of the EIC peak area after repeated injection of CORT for 10 times with the flow rate of 0.3 and 0.6 mL/min separately with the same gradient. Predominant presence of hydrogen ion adduct [M þ H]þ for CORT (m/z 347.2217) was observed in both ionization modes with the retention time of 6.5 and 4.2 min for ESI and APCI separately. The signal produced with APCI was nearly three times stronger than ESI.

Optimization of ion source parameters To determine the optimum, the fragmentor voltage was investigated from 60 to 175 V separately and the intensity of EIC peak area was shown in Figure 2. When the fragmentor was ,120 V, signal increased as voltage, then reduced when the fragmentor .120 V. Suitable fragmentor voltage was obtained at 120 V. Vaporizer assists the evaporation of the solvent in the source of APCI. Most of the heat is expected to be consumed for the evaporating of the sheath liquid and heating of the nebulizer gas. In our experiments, when the temperature was set between the range from 200 to 3008C improved the evaporation of the solvent with altering spray characteristics through the reduction in droplet diameter and size and thus increased the response and improved reproducibility (reduce relative standard deviation, RSD). A significant degradation of CORT was obtained when the temperature .3008C (Figure 3I). However, the vaporizer temperature was set at 3508C for an increased precision. Drying gas temperature varying 200, 250, 300, 350 and 4008C was investigated in this paper, and it had no great impact on the sensitivity (Figure 3II). However, enhanced precision was obtained when drying gas temperature was 4008C. For the

Results Optimization of MS conditions Sensitivity and reproducibility are essential characteristics for quantitative analysis that was often ignored in application of qualitative analysis with Q-TOF MS. Efforts were made to optimize the

Figure 2. Optimization of fragmentor.

An Optimized Method for CORT Analysis 3

enhanced precision, vaporizer was 3508C with the drying gas 4008C.

Optimization of MS parameters In order to obtain the best quantitative data, scan rate should be optimized with fixed liquid flow (11, 12). We compared the scan rate of 1, 2, 3, 4 and 5 spectra/s separately for data acquisition in centroid mode with the flow rate of 0.6 mL/min. As shown in

Figure 4, elevated scan speed significantly increased the number of data points taken across a peak and significantly decreased the response with enormous data files. When scan rate was 1 spectra/s, intervals between the adjacent points were 0.016 – 0.017 s with 11 points across a peak. There were .50 points across a peak when the scan rate increased to 5 spectra/s. With the scan rate of 2 spectra/s, 15 – 18 data points were obtained across the peaks which were more than sufficient for quantitation.

Figure 3. EIC and RSD of changes with temperatures: I: changes with vaporizer temperature and II: changes with gas temperature.

Figure 4. EIC peak areas versus scan rate and the points in each peak.

4 Li et al.

Mass extraction window EICs of CORT and IS generated with a specified MEW were used for calculation after acquisition of full-scan data. MEW is critical in LC–HRMS for quantitative analysis for that a wide MEW could introduce unwanted interference or higher background by extracting response from compounds with similar m/z’s to the analyze along with the analyte of interest while a very narrow MEW could increase the probability of reporting a false negative at low concentrations. Jemal (12) had compared the matrices with different MEWs at the retention time of the analytes; however, as an endogenous compound the blank biological matrices were difficult to obtain for CORT detection. For our experiment, we compared 10 –500 ppm for CORT quantification and negligible back ground noise was observed with a relatively wide MEW of 50 ppm. Additionally, we did not observe any pernicious effect on the accuracy and precision of the method, even at low concentrations. This was likely attributed to the liquid-liquid extraction method and LC gradient, which made we were able to separate the impurities from the analytes.

Evaluation of performance of LC– HRMS method Validation of this method was carried out using calibration standards and QC samples. In this method, selectivity, recovery and sample stability were involved in validation procedures.

Selectivity and matrix effect Centroid mass spectra of TIC and EIC of charcoal-treated pooled plasma (I), true plasma with IS (II) and spiked true plasma with IS (III) were shown in Figure 5. A satisfactory separation on endogenous impurities and CORT was obtained with a gradient elution with the retain time of 4.20 min for CORT and 2.56 min for IS. No interfering peak was observed at the retention times of both compounds. The predominant presence of hydrogen ion adduct [M þ H]þ for CORT (m/z 347.22) as well as IS (m/z 359.18) was detected separately. Charcoal was used to eliminate the inherent effect of endogenous CORT in plasma in this paper. Figure 5I showed that charcoal absorbed all of the CORT with the content of 20%. CORT in plasma showed the same retention time and m/z features with spiked sample. In order to verify selectivity of the assay, the eluent corresponding to the 4.2 min peak with m/z 347.2217 (+0.5 m/z) was collected from methanolic solution and plasma samples and subjected to MS analysis with the collision energy of 18 V (Figure 6). Fragment ions of CORT in methanol solution were similar to the ions in plasma sample. The protonated molecular ion of CORT at m/z 347.22 by loss of a successive neutral loss a molecule of H2O (molecular weight ¼ 18) gave the major fragment ion at m/z ¼ 329.2117 and 311.2006 was the main ion fragments with the deviation of ,50 ppm. Fragmentation pattern of CORT was described in Figure 7. The matrix effect of CORT at 16.48, 82.4 and 412 ng/mL was 91.0 + 0.12, 94.5 + 0.05 and 97.4 + 0.09%, respectively. The matrix effect of IS was evaluated in a similar way, and the result was 104.7% (CV , 10%). The result indicated that a slight inhibition interfered with the ionization of the CORT and a slight enhancement occurred for the IS.

Precision, accuracy and recovery The CV for within-run precision was 5.6% for 16.48 ng/mL, 3.2% for 82.4 ng/mL and 2.5% for 412 ng/mL. The CV for between-run precision was 6.9% for 16.48 ng/mL, 5.1% for 82.4 ng/mL and 3.1% for 412 ng/mL, respectively. The mean accuracy for CORT observed during a within-run experiment was 7.4% for 16.48 ng/ mL, 5.6% for 82.4 ng/mL and 23.22% for 412 ng/mL with the RE ranged from 4.4 to 6.2%. The percentage accuracy for betweenrun analysis ranged from 25.9 to 8.6% with an RE of 7.9–10.6%. Recoveries were determined from plasma sample and spiked plasma samples. Plasma samples were mixed with standard solutions of CORT and extracted as described above. Plasma extracts were prepared, and a portion of the final extracts were prepared and a portion of the final extracts were mixed with standard solutions of CORT. The recoveries in the range of 8.24– 412 ng/mL were shown in Table I.

Linearity Q-TOF LC–MS has a relatively narrow linear dynamic range. It has been our experience that APCI response levels out even at nanogram concentrations leading to calibration plots which vary from compound to compound and even for the same compound depending upon MS conditions. In the present study, we observed a marked deviation in the straight line plot of .412 ng/mL on column injections (1,030 and 2,060 ng/mL data were not shown). Therefore, the linearity of the method was investigated in the concentration range of 8.24 –412 ng/mL. The representative regression equation was y ¼ 0.0056x þ 0.003. The value of R 2 was 0.9998. The LOD was 1.696 ng/mL, and the LOQ was 5.088 ng/mL with S/N 3 and 10, respectively. Representative chromatograms were shown in Figure 8.

Sample stability CORT was stable in plasma for 4 h at room temperature (recoveries 90.532102.11%) and for 30 days at 2808C (recoveries 97.102100.16%). No significant deviation was found from the nominal values. At least three freeze–thaw cycles can be tolerated without losses higher than 15%. The values were given in Table II.

Application to evaluate incurred samples This validated analytical method was used to determine concentrations of CORT in plasma samples from the stressed mice with and without ginseng treatment for the stress state estimation. The representative chromatogram of the intended sample was illustrated in concentrations calculated using the peak area ratio of the endogenous analyte to IS, and the corrected regression equation of the calibration curve derived from the surrogate analyte. Concentrations of CORT in plasma obtained from normal, stressed and ginseng-treated stressed mice were shown in Figure 9. The endogenous concentration of CORT in the stressed group was markedly higher than that of the normal group, rising from 44.02 + 8.71 to 157.02 + 36.08 ng/mL. Ginseng significantly alleviated the increase of CORT induced by immobilization stress with the concentration of 106.99 + 23.41 ng/mL. An Optimized Method for CORT Analysis 5

6 Li et al.

Figure 5. TIC spectra of the three samples (A) and shows EIC spectra of the three samples (B).

Figure 6. Product ion spectra of precursor ion with m/z 347.22. (A) 50 ng/mL methanolic solution of CORT. (B) Plasma sample of with CORT with the concentration of 40 ng/mL.

Figure 7. Fragment pattern of CORT.

Discussion Table I Recoveries of CORT (n ¼ 5) Concentration (ng/mL)

Measured

Recovery (%)

16.48 82.4 412

18.46 + 9.35 84.87 + 5.76 396.38 + 4.01

112.01 + 8.2 102.99 + 7.0 96.20 + 6.4

Unlike LC–MS-MS methods, which require identifying the product ions and their optimal collision energies and the source parameters such as gas flow rate and temperature, LC – HRMS methods, in general, only require optimizing the source parameters. EICs on individual compounds can then be obtained postdata acquisition (11).

An Optimized Method for CORT Analysis 7

Figure 8. EIC of LOD and LOQ samples.

Table II Stability Studies on CORT in Mouse Plasma (n ¼ 5) Concentration (ng/mL)

QC1(16.48) QC2(82.4) QC3(412)

Short-term stability

14.92 + 0.42 80.96 + 2.52 420.72 + 13.84

Long-term stability

15.48 + 1.55 82.54 + 5.63 400.8 + 18.32

Figure 9. Plasma CORT concentration (ng/mL) in normal, stressed and ginseng-treated mice. Data in each group represent the mean + SEM analyzed by analysis of variance; *P , 0.05 compared with normal group; #P , 0.05 compared with stressed group.

ESI is a process that transfers ions from the liquid into the gas phase through ion desorption from droplets, while APCI is an ion source that phase is evaporated in a heated region of the ion source, and the analytes are ionized through gas-phase reactions in the corona discharge region. It is generally accepted that APCI is suitable for the ionization of compounds with poor 8 Li et al.

Aut-sampler stability

17.92 + 1.77 81.06 + 5.21 407.08 + 30.57

Freeze –thaw stability Cycle 0

Cycle 3

15.92 + 1.57 82.45 + 6.59 418.53 + 35.24

14.99 + 1.04 83.07 + 7.92 381.62 + 21.52

polarity. However, a search of the literature yielded no reports comparing these two methods in terms of ionization efficiency for CORT or other glucocorticoids. In present study, the stronger response signal was also attributed to the broadening of peaks caused by the lower flow rate tolerance in ESI than APCI. Fragmentor voltage was the most important parameter affecting fragmentation and transmission of sample ions. In APCI, the fragmentor voltage gives ions a “push” that helps their passage through the relatively high-pressure region between the exit of the capillary and the skimmer. Generally, a higher fragmentor voltage could induce more fragmentation to occur and results in better ion transmission for compounds that do fragment easily. Provided the analyte was thermolabile and did not undergo degradation when passing the short heated zone of the vaporizer, vaporizer temperature was a critical parameter. It had a large impact on signal response and sensitivity that should be optimized in quantitative analysis. For robust data acquisition and quantitation in the centroid mode, scan rates of 1 – 5 spectra/s were compared with the flow rate of 0.6 mL/min to insure that a sufficient number of data points were collected across the chromatographic peaks. Elevated scan rates significantly increased the number of data points obtained across a peak and significantly decreased the response by generated an enormous amount of data. Feasibility was evaluated with the scan rate of 2 spectra/s and provided 15 –18 data points were obtained across the peaks which were more than sufficient for quantitation. It should be noted that when a higher LC flow rate is desired for fast chromatography, users should optimized the scan rate to insure a sufficient number of data points are collected.

Table III Analysis of CORT by Various Detection Methods Specimen

Mouse blood Mouse blood Human serum Human serum

CORT (ng/mL)

Method

Non-stressed

Stressed

Ionization

Mass analyzer

Mass detection

LOD (ng/mL)

Reference

44 8 – –

157 176 – –

APCIþ ESIþ APCIþ ESIþ

TOF Quadrupole Quadrupole Quadrupole

EIC MRM MRM MRM

1.696 0.3 1 1.15

In this study (21) (15) (22)

APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; CORT, corticosterone; LOD, limit of detection; MRM, multiple reaction monitoring.

After full-scan data were acquired, EICs for each of the analytes and their IS were generated with a specified MEW for all samples. A narrower MEW may be necessary for cruder extracts obtained from poor separation the analytes and impurities without ion suppression or enhancement. It is therefore important to carefully evaluate the effect of MEW during method development, and during the sample analysis phase. It may affect the accuracy and precision of the quantitation, lead to interfering endogenous peaks, or result in false negatives at low concentrations. Q-TOF MS analysis of CORT was associated with several challenges. Many structurally similar steroids and metabolites were present at low concentrations in biological samples, particularly, for animal experiments. Determination of CORT required high specificity, good accuracy and sufficient precision for both analytes. LC– MS technology could be considered the most appropriate method to satisfy all the requirements for simultaneous detection. Table III summarizes some analytical data from different biological samples using various detection methods. LC – HRMS with TOF was as sensitivity as quadrupole. This represents the successful detection of CORT from blood samples by Q-TOF LC – MS technology. This is an extremely useful and important finding will be of great interest to investigators who are interested in applying LC – HRMS to quantitative analysis especially for the analysis of complex biological samples. Plasma CORT level in mouse can be provoked by a number of stressors that include electrical shock and tail clipping. Excluding chemical stimulation, immobilization is a simple physical method that can be used for inducing stress. In present study, mice were administered with saline or ginseng saponin orally for 7 consecutive days to investigate the protective effect of ginseng against stress. Although this type of intervention is considered mild, the act of handling and gavaging could cause sufficient stress in mice. This could explain why plasma CORT concentrations were higher in our study than the ranges reported in other literatures.

Conclusion The use of a full-scan LC–HRMS assay demonstrated in this paper satisfied validation acceptance criteria in terms of accuracy, precision and selectivity commonly adopted for the LC– MS approach. Therefore, it is feasible to use LC –HRMS for quantitative analysis in regulated bioanalysis. Before LC–HRMS is fully implemented and integrated in regulated bioanalysis, additional research is necessary. The effect of MEW and resolution on the accuracy and precision of the method should be investigated.

Further improvements in sensitivity, acquisition speed and the quantity of data generated are other necessary areas of focus.

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An optimized method for corticosterone analysis in mouse plasma by ultra-performance liquid chromatography-full-scan high-resolution accurate mass spectrometry.

A simple method based on liquid chromatography-full-scan high-resolution accurate mass spectrometry (LC-HRMS) using a quadrupole time-of-flight (TOF) ...
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