Research Article Received: 3 February 2014

Revised: 12 March 2014

Accepted: 21 March 2014

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 1293–1302 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6902

The application of a new microfluidic device for the simultaneous identification and quantitation of midazolam metabolites obtained from a single micro-litre of chimeric mice blood Richard Gallagher1*, Leonard Dillon3, Aidan Grimsley1, Jim Murphy4, Kristin Samuelsson2 and David Douce3 1

Oncology iMed DMPK, AstraZeneca UK Ltd., Alderley Park, Macclesfield, Cheshire SK10 4TG, UK AstraZeneca DMPK, Pepparedsleden 1, 43183, Molndal, Sweden 3 Waters Corp., Floats Road, Manchester M23 9LZ, UK 4 Waters Corp., 34 Maple Street, Milford, MA, USA 2

RATIONALE: Improvements in the design of low-flow highly sensitive chromatographic ion source interfaces allow the detection and characterisation of drugs and metabolites from smaller sample volumes. This in turn improves the ethical treatment of animals by reducing both the number of animals needed and the blood sampling volumes required. METHODS: A new microfluidic device combining an ultra-high pressure liquid chromatography (UHPLC) analytical column with a nano-flow electrospray source is described. All microfluidic, gas and electrical connections are automatically engaged when the ceramic microfluidic device is inserted into the source enclosure. The system was used in conjunction with a hybrid quadrupole-time-of-flight mass spectrometer. RESULTS: The improved sensitivity of the system is highlighted in its application in the quantification and qualification of midazolam and its metabolites detected in whole blood from chimeric and wild-type mice. Metabolite identification and full pharmacokinetic profiles were obtained from a single micro-litre of whole blood at each sampling time and significant pharmacokinetic differences were observed between the two types of mice. CONCLUSIONS: Improvements in the enhanced ionisation efficiency from the microfluidic device in conjunction with nanoUHPLC/MS was sufficiently sensitive for the identification and quantification of midazolam metabolites from a single micro-litre of whole blood. Detection of metabolites not previously recorded from the chimeric mouse in vivo model was made. Copyright © 2014 John Wiley & Sons, Ltd.

The miniaturisation of chromatographic techniques for use in conjunction with mass spectrometry (MS) is a highly active research area.[1–7] This technology, often referred to as microfluidics or ’lab-on-a-chip’, was first reported in 1979 for a gas chromatography chip based system.[8] Liquid chromatography (LC) devices were later developed during the 1990s, first for capillary electrophoresis/electro-chromatography[9–13] and later for electrospray ionisation mass spectrometry.[14,15] More recently, the coupling to matrix-assisted laser desorption ionisation has been reported.[16] Commercial microfluidic devices for LC/MS applications often comprise of a trapping column, an analytical column and an electrospray ionisation emitter all combined into a single chip design.[17–19] The trapping column allows for the clean up or concentration of a sample before switching it to the analytical column.[20] These systems have mainly used nano-litre flow rates, taking advantage of the improved ionisation efficiencies obtained at these reduced flow rates.[21]

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* Correspondence to: R. Gallagher, AstraZeneca, Mereside 24 F87, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. E-mail: [email protected]

Nano-ultra high-performance liquid chromatography (nanoUHPLC) benefits from high separation efficiencies and resolution obtained from using smaller column dimensions and packing materials and is ideal for integration into a microfluidic device.[22–25] For example, decreasing the internal diameter of the column from a conventional size of 2.1 mm to 150 μm effectively increases the eluting peak concentration by 196-fold.[26] Also in this low-flow LC technique the critical transfer of the dissolved molecules into the gas phase benefits from the higher ionisation efficiency and reduced droplet sizes produced from smaller nano-flow electrospray emitters. Other advantages compared to conventional chromatography systems are better long-term ion source performance, as smaller sample volumes are injected and significantly reduced eluent volumes are required for the chromatographic analysis. The coupling together of nano-electrospray and UHPLC into a micro-fluidic device will have many analytical applications where only small sample quantities are available. Such an example of this is the analysis of plasma samples obtained from chimeric mice in vivo models. Drugs and other xenobiotics entering the body are generally subject to metabolism that facilitates their detoxification and elimination. The ability to predict the metabolic fate of compounds

R. Gallagher et al. before the first doses are given to humans is highly desirable for reasons of both efficacy and safety. In the chimeric mouse in vivo model, the liver of the mouse has been humanised through the replacement of their hepatic cells with human hepatocytes.[27–30] In the chimeric mouse (Genotype: uPA+/+/SCID (severe combined immunodeficient), denoted as PXB) the mouse liver cells have been replaced with human cells to around 70–80%. Potentially, these mice can be used to assess significant human metabolites of a drug candidate and their related hepatic effects early in the drug development program. However, the size of the mice restricts the sample volume that can be safely taken during a pharmacokinetic time study for the drug and its metabolites. Therefore, an analytical system capable of producing the maximum data from a small sample volume is highly desirable to maximise both scientific information and reduce animal usage. The following sections will describe the identification and quantification of midazolam and its predominant human metabolites from a single micro-litre of whole blood obtained from PXB and the control SCID mice utilising the high ionisation efficiency of the prototype microfluidic nanoUHPLC column and emitter. The samples analysed in this study were generated in a previous study where pharmacokinetic profiles of the parent compound as well as 1’-hydroxymidazolam were established from five microlitre (5 μL) blood sample volumes.[29] In this study we reveal the profiles of two more metabolites, 4’-hydroxymidazolam and the secondary metabolite 1’-hydroxymidazolam glucuronide from a single micro-litre of blood. In addition to the pharmacokinetic profiles further metabolites were observed in this study using samples from mice dosed at levels 10 times lower (1 mg/kg) to those used in the previously reported work (10 mg/kg).[29]

EXPERIMENTAL The assay is based on the direct analysis of extracts from whole blood produced by protein precipitation and subsequent LC/MS analysis on a nanoUHPLC system with all chromatographic separations occurring on a ceramic microfluidic device. The ion source of the mass spectrometer incorporates the ceramic microfluidic device which contains a 5 cm analytical column of 150 μm diameter along with the ionisation emitter in one interchangeable unit. All quantitation and structural elucidation was performed on a hybrid quadrupole-time-of-flight (Q-tof) mass spectrometer operating in MSE data-acquisition mode.

dimethyl suldoxide (DMSO), leucine enkephalin, methanol and formic acid were supplied by Fisher Scientific UK Ltd. (Loughborough, UK). Solutions and standards Three stock solutions at 1 mg/mL in DMSO were made for each of the analytes (midazolam, 1’-hydroxymidazolam, 4’-hydroxymidazolam and 1’-hydroxymidazolam-O-glucuronide). An aliquot from each stock solution was diluted to 0.1 mg/mL in methanol and the absorbances of the substrates were measured at 285 nm. The analyte concentrations in the three stock solutions were within a 5% variance for each analyte and two different stock solutions of each analyte were used to prepare the standards and the quality controls samples (QCs), respectively. The stock solutions of the four analytes were mixed and a series of dilutions in methanol were made. A 5 μL aliquot of methanol standard was spiked into 95 μL of control blood to generate six standards with the nominal concentration in blood of 0.5, 1, 25, 100, 500 and 750 ng/mL. QCs were prepared in the same way as the standards using a different set of stock solutions and the nominal concentrations in blood constituting the QCs were 25, 50, 100 and 500 ng/mL. Pharmacokinetic study Samples analysed in this study were generated from the 1 mg/kg dose of midazolam described in Samuelsson et al.[29] The study was approved by the Animal Ethics Committee at PhoenixBio Co. Ltd. and the in life phase of the study was conducted at PhoenixBio Co. Ltd. Briefly, two SCID mice sourced from CELA Ltd. (Higashi-Hiroshima, Japan) and two chimeric PXB mice with humanised livers, generated at PhoenixBio Co. Ltd. by the method described previously, were dosed orally with midazolam (1 mg/kg).[27] Blood samples (15 μL) were collected from each animal via the retro-orbital sinus/plexus using calibrated pipettes containing K2EDTA (Drummond Scientific Co., PA, USA) under isoflurane (Escain®, Mylan, Osaka, Japan) anaesthesia at the following time points post-dosing; 0.083, 0.167, 0.5, 1, 2, 3, 8, 12, and 24 h. Sample preparation for qualitative and quantitative analysis The internal standard 2H4-1’-hydroxymidazolam was dissolved in water to a concentration of 0.5 μg/mL. The 1 μL aliquots of blood, sample standards or QCs were extracted by the addition of 10 μL of acetonitrile and then vigorously mixed before centrifugation at 30 000 rpm for 2 min. A volume of 7 μL of the supernatant was transferred to a 96-well plate which contained 10 μL of internal standard solution in each well.

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Chemicals

Microfluidic device and nanoUHPLC

Midazolam was acquired from Apin Chemicals Ltd. (Oxford, UK) and authentic metabolite standards for 1’-hydroxymidazolam, 4’-hydroxymidazolam and 1’-hydroxymidazolam-O-glucuronide were procured from Onyx Pharmaceuticals Inc. (San Fransisco, CA, USA), Sigma-Aldrich (Schnelldorf, Germany) and Carbosynth Ltd. (Compton, UK), respectively. 2H4-1’-Hydroxymidazolam was purchased from LGC Standards (Teddington, UK) and used as an internal standard for pharmacokinetic analyses. Acetonitrile,

The prototype microfluidic device used for all the chromatography and electrospray analysis consists of a ceramic tile in which a 5 cm × 150 μm i.d. column is etched. The column is packed with sub-2 μm particle BEH C18 stationary phase (Waters Corp., Milford, MA, USA) and can be temperature controlled via an integrated heater and thermocouple. The column is connected directly to a metal nebulised electrospray emitter (35 μm i.d.). The metal capillary has a higher

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Microfluidic device for identification and quantitation of metabolites hydrophobicity than silica resulting in a reduced residence at the capillary tip and aiding in the spray formation. The capillary is produced through acid etching and the tip is passivated to ensure an inert surface. The microfluidic device is inserted directly into a Trizaic nanoTile™ ionisation source (Waters Corp.) where all microfluidic, gas and electrical connections are automatically engaged. The device dimensions are approximately 12 cm × 4 cm × 0.8 cm and a diagram of the device is shown in Fig. 1. Chromatography was performed using a Waters nano Acquity Sample Manager and Solvent Manager (Waters Corp.). Separations on the prototype microfluidic ceramic tile were performed using reversed-phase gradient chromatography. The column temperature was set to 30 °C and the eluent from the column flowed directly to an incorporated nebulised ESI capillary located on the microfluidic device. An eluent flow rate of 1.2 μL/min was used and 3 μL of sample extract were injected onto the integrated UHPLC column. The chromatography eluents used were: aqueous mobile phase A (0.1% (v/v) formic acid) and organic phase B (100% acetonitrile + 0.1% (v/v) formic acid). The initial conditions for the gradient consisted of 10% solvent B (held for 4 min), which was increased to 90% B over a period of 3 min and maintained for 11 min before returning to 10% solvent B. Column equilibration for 3 min was performed between injections. Mass spectrometry analysis Mass spectrometry was initially performed using a Waters Xevo G2 Q-tof mass spectrometer with all the quantitative results completed on a Waters Xevo G2-S Q-Tof mass spectrometer both fitted with a Trizaic nanoTile™ ionisation source operating in positive ion mode (Waters Corp.). The mass spectrometers performed alternative high- and lowenergy scans, known as the MSE acquisition mode. The detection and quantification of analytes were performed using extracted accurate mass chromatograms (5 ppm mass window). Parameters that were kept constant for all the analytes included the capillary voltage (3.6 kV), cone voltage (20 V) and the source temperature (maintained at 120 °C). The cone gas flow rate was 50 L/h, nano-flow gas pressure set at

0.3 bar and the collision gas flow rate was set to 0.15 mL/min. The two mass spectrometers differ in that the Xevo G2-S Q-Tof is fitted with an additional ion optical device called the StepWave™ which is a differentially pumped off-axis lens system designed to efficiently capture ions and remove neutral material entering the mass spectrometer.[31] This increased ion sampling efficiency and noise removal produces a significant improvement in the limits of detection (see Discussion section). The data were lock mass corrected using leucine enkephalin (MH+ 556.2771 Da). All of the analyses were conducted at a resolution of approximately 22 000 at full width half maxima (FWHM). The MS analyses of the whole blood extracts were completed using ESI + mode. The acquisition rate was 0.1 s with an inter-scan delay of 0.01 s over a mass range of m/z 50–1200. In all cases a 3 μL injection of the aqueous extract was injected into the NanoAcquity MS system. All instrument and data acquisition parameters were controlled by MassLynx™ (version 4.1; Waters Corp.) and the data generated was processed using a beta test version of UNIFI™ software (version 1.6; Waters Corp.). Data analysis and method validation All quantitative data was processed using TargetLynx™ contained within the MassLynx™ control software. Calibration curves were constructed using linear regression with a 1/x weighting of the peak area ratios (analyte/internal standard) versus the nominal concentration for the calibration standards. The analysis was initiated by injecting the standards followed by sample analysis from the four mice. The QCs were analysed in between each set of mouse samples making four replicates of each QC. Two blanks were injected between each set of samples and QCs. The criteria used for acceptance of the run were that the average values obtained for the QCs measured were within 30% of the nominal concentration allowing exclusion of no more than one QC sample, at each level and analyte of those injected. Each calibration standard used to construct the calibration curve had to be within 25% of the nominal concentration. All the standards were used to construct the calibration line except for the 1-OH glucuronide MDZ which had some missed injections resulting in only 50% of the calibration points being used (75 to 750 ng/mL). The accuracy was evaluated by comparing the mean measured concentrations of the QCs with their nominal concentrations and the precision for each QC level was calculated by dividing the standard deviation of the measured values by the mean of the measured values. The analysis was deemed acceptable for the samples if accuracy and precision were within 30%. The lowest calibration standards of midazolam and 1’-hydroxymidazolam, 4’-hydroxymidazolam and 1’-hydroxymidazolam-O-glucuronide were used to define the limits of quantification (LOQs) for the analytes. The limits of detection of these analytes were not formally assessed. Pharmacokinetic data analysis

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Non-compartmental pharmacokinetics were calculated using WinNonlin 5.2 (Pharsight, Mountain View, CA, USA). The area under the curve from 0 to 3 h (AUC0–3h) was estimated using the linear trapezoidal rule. Pharmacokinetic parameters are reported as means of two animals per group and dose level.

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Figure 1. Schematic diagram of the prototype ceramic microfluidic device. (1) Metal electrospray emitter. (2) Capillary voltage connection pad. (3) Post-column flow. (4) Ceramic plate. (5) Electrical connectors (e.g. column heater, thermocouple). (6) Infusion tile flow route. (7) Alignment port. (8) Waste. (9) LC flow to column. (10) BEH C18 UHPLC column (50 mm × 150 μm i.d.). (11) Sheath gas inlet.

R. Gallagher et al. Table 1. Intra-assay accuracy and precision (%) of quality control samples

Midazolam

Conc. (ng/mL) 25 50 100 500

1’-Hydroxymidazolam

4’-Hydroxymidazolam

1’-HydroxymidazolamO-glucuronide

Accuracy

Precision

Accuracy

Precision

Accuracy

Precision

Accuracy

Precision

0.6 12.3 2.2 3.3

19.4 20.6 9.3 8.1

2.6 11.4 13.2 –0.8

16.4 12.7 6.7 9.8

–13.5 –13.0 5.2 1.4

16.8 3.0 16.3 6.0

–7.1 14.4 20.0 17.5

46.0 19.9 10.0 11.5

Table 2. Mean pharmacokinetic parameters for midazolam and its metabolites in SCID and PXB mice after oral administration of midazolam (1 mg/kg) AUC0-3h (ng × h/mL)

SCID PXB

n

MDZ

1’OH

4’OH

1’OH-Gluc

2 2

6.3 27.5

95.7 42.9

1.6 5.3

81.6 1639.0

RESULTS Midazolam metabolite quantification All four previously identified analytes were quantified using linear calibration lines produced from each compound using reconstructed ion chromatograms of the expected [M + H]+ ion mass/charge ratio for midazolam, 1’-hydroxymidazolam, 4’-hydroxymidazolam and 1’-hydroxy-O-glucuronide using the TargetLynx™ software (5 ppm mass window). The accuracy and precision of QC samples were within ±20.0% and ±20.6%, respectively, except for the lowest QC of 1’-hydroxymidazolam glucuronide which had a precision value of 46.0% (Table 1). To compare the two mouse strains the data for each set was averaged and a summary of the pharmacokinetic parameters of midazolam and its metabolites in PXB and SCID mice following an oral dose of 1 mg/kg is presented in Table 2. Major differences in the exposure of both midazolam and its metabolites are readily seen with higher mean AUC0–3h values of midazolam (27.5 and 6.3 ng × h/mL), 4-hydroxymidazolam (5.3 and 1.6 ng × h/mL) and 1-hydroxymidazolam glucuronide (1639.0 and 81.6 ng × h/mL) in the PXB mice compared to the control SCID mice. In the latter case a 20-fold increase in glucuronide is measured. The AUC0–3h of the 1-hydroxymidazolam metabolite was 2-fold the magnitude in the SCID mice (95.7 ng × h/mL) compared to the PXB mice (42.9 ng × h/mL). Midazolam metabolite identification

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Accurate mass chromatograms were used to extract midazolam and its metabolites from the raw data; Fig. 2 illustrates data from a 10 min PXB mouse sample. The stability of the system was such that the retention time shift across all samples was less than 0.01 min. Midazolam was detected in all samples (326.0851 Da, –1.165 ppm error to

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Figure 2. Extracted ion chromatograms obtained from the PXB strain mouse, T = 10 min sample. Chromatograms (top to bottom): (a) midazolam (326.086 Daltons (Da)), (b) 1’- and 4’-hydroxymidazolam (342.081 Da, Rt. = 13.25 and 13.18 min, respectively), (c) 1’-hydroxymidazolam glucuronide (528.113 Da), and (d) the internal standard 2H41’-hydroxymidazolam (346.105 Da). The mass windows on the chromatograms are 10 ppm.

theoretical value) at a retention time (Rt.) of 13.29 min. In the detection of putative metabolites three criteria were required to be satisfied before a compound was deemed identified: (i) the molecular ion isotope region showed the characteristic isotope pattern for incorporation of a chlorine atom, (ii) the mass measurement accuracy of the protonated molecular ion [M + H]+ was within 2 ppm of the theoretical value for the proposed structure, and (iii) the metabolite product ions (also measured with 2 ppm) were consistent with the proposed structure and/or seen in the reference standards. Following these criteria the subsequent metabolites were detected and are listed in Table 3. An example of spectral quality is shown in Fig. 3 for 1’-hydroxymidazolam-Oglucuronide and putative product ion structures are shown in Fig. 4 for midazolam. The metabolites detected are listed below:

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Microfluidic device for identification and quantitation of metabolites Table 3. Summary of the midazolam metabolites detected via nano-flow UHPLC-MSE and their corresponding diagnostic product ions. Accurate mass measured for all fragments was within 2 ppm of the theoretical value of the proposed elemental composition Measured [M + H]+ (Error) Elemental composition Peak ID Rt. (min)*

Assignment

MSE product ions (Da)

Elemental composition of product ion

Parent

13.23

326.0851 Da (–1.165 ppm) C18H14N3ClF

Midazolam (MDZ)

291.1164 249.0827 244.0328 223.0796 209.0638 129.0557

C18H14N3F C16H10N2F C14H8NClF C15H10NF C14H8NF C9H7N

M1**

13.19

342.0799 Da (–1.446 ppm) C18H14N3OClF

1’-Hydroxy-MDZ

324.0705 297.0618 262.0898 203.0375 168.0686

C18H12N3ClF C17H11N2ClF C17H11N2F C11H8N2Cl C11H8N2

M2**

13.11

342.0803 Da (–0.276 ppm) C18H14N3OClF

4’-Hydroxy-MDZ

325.0546 297.0586 234.0484 122.0399 109.0396

C18H11N2OClF C17H11N2ClF C13H10NClF C7H5NF C5H5N2O

M3**

13.02

358.0751 Da (–0.584 ppm) C18H14N3O2ClF

1’,4’-Dihydroxy-MDZ

341.0483 340.0648 313.0538 262.0900 234.0480

C18H11N2O2ClF C18H12N3OClF C17H11N2OClF C17H11N2F C13H10NClF

M4

13.19

360.0913 Da (0.946 ppm) C18H16N3O2ClF

MDZ + H2O2

331.0645 315.0699 301.0541 287.0745 273.0594 249.0589

C17H13N2O2ClF C17H13N2OClF C16H11N2OClF C16H13N2ClF C15H11N2ClF C13H11N2ClF

M5**

13.01

518.1119 Da (–1.124 ppm) C24H22N3O7ClF

1’-hydroxy-O-glucuronide MDZ

342.0800 324.0699 297.0618 203.0372

C18H14N3OClF C18H12N3ClF C17H11N2ClF C11H8N2Cl

M6

13.32

358.0749 Da (–1.143 ppm) C18H14N3O2ClF

Dihydroxy-MDZ

340.0644 309.0584 291.0799

C18H12N3OClF C18H11N2ClF C17H10N3OF

M7

13.08

502.1185 Da (1.857 ppm) C24H22N3O6ClF

N-Glucuronide

326.0858

C18H14N3ClF

M8

13.16

376.0856 Da (0.728 ppm) C18H16N3O3ClF

MDZ + H2O3

Non-reliable detection

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*Retention time (Rt.) all taken from SCID mouse except M2 and M7 obtained from PXB mouse. **Compared to authentic reference standard.

R. Gallagher et al.

Figure 3. Example of mass spectrum, MS and MSE, obtained for 1’-hydroxy-O-glucuronide MDZ.

• M1, Rt. 13.19 min, exhibits a protonated molecular ion [M + H]+ at 342.0799 Da and the accurate mass measurement confirmed this to correspond to the addition of a single oxygen atom (–1.165 ppm error). The retention time and product ions detected in the high-energy MSE scan of M1 were in accordance to the reference standard 1’-hydroxymidazolam. Characteristic product ions nominally at 168, 203, 262, 297 and 324 Da, the latter formed by simple loss of water, and 297 Da formed by loss of water and HCN.

• M2, Rt. 13.11 min. The retention time, molecular weight (342.0803 Da, –0.276 ppm error) and product ions detected were in accordance to the reference standard 4’-hydroxymidazolam. Characteristic product ions nominally at 109, 122, 234, 297 and 325 Da, the latter formed by loss of ammonia, and 297 Da formed by loss of water and HCN. • M3, Rt. 13.02 min, [M + H]+ at 358.0751 Da. Accurate mass measurement confirmed this to correspond to the addition of two oxygen atoms (–0.584 ppm error). M3 showed the major loss to be NH3, H2O and the product ion 234 Da (seen for M2). This is consistent with oxidation at both the 1’- and 4’-position. • M4, Rt. 13.19 min, [M + H]+ at 360.0913 Da. Accurate mass measurement confirmed this to correspond to the addition of two oxygen and two hydrogen atoms (0.946 ppm error). A characteristic product ion nominally at 273 Da that is consistent with all the biotransformation occurring on the methyl-imadazo-diazepine rings structure. The exact nature of the metabolite is unknown but could for example be a ring-opened species, 4’-hydroxymidazolam with water addition or a dihydro addition across a double bond. • M5, Rt. 13.02 min. The retention time, molecular weight (518.1119 Da, –1.124 ppm error) and product ions detected were in accordance to the reference 1’-hydroxymidazolamO-glucuronide. The highest value m/z product ion is nominally at 342 Da corresponding to 1’-hydroxymidazolam formed from loss of glucuronic acid which subsequently loses water to form the most intense product ion in the spectrum at 324 Da. • M6, Rt. 13.32 min, [M + H]+ at 358.0749 Da. Accurate mass measurement confirmed this to correspond to the addition of two oxygen atoms (–1.142 ppm error). M6 showed major product ions nominally at 291, 309 and 340 Da but the fragmentation information was insufficient to assign the positions of oxidation. • M7, Rt. 13.08 min, [M + H]+ at 502.1185 Da. Accurate mass measurement confirmed this to correspond to the addition of a single glucuronic acid moiety to MDZ (1.857 ppm error) and the product ion spectrum obtained is consistent with the proposed structure being

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Figure 4. Fragmentation assigned to observed product ions in MSE mass spectra for midazolam [M + H]+ 326 Da. Accurate mass measured for all fragments was within 2 ppm of the theoretical value of the proposed elemental composition.

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Microfluidic device for identification and quantitation of metabolites midazolam-N-glucuronide. There are potentially three isomeric forms of this metabolite as the glucuronic acid can attach to any of the three nitrogen atoms. The product ion mass spectrum shows direct loss of the glucuronic acid to form a product ion nominally at 326 Da but no other useful structural information could be obtained to confirm its point of attachment to MDZ. • M8, Rt. 13.16 min, [M + H]+ at 376.0856 Da. Accurate mass measurement confirmed this to correspond to the addition of three oxygen and two hydrogen atoms (0.728 ppm error). This particular metabolite eluted too close to the internal standard for reliable fragment ions to be identified as being unique to this compound. However, the isotope pattern shows the incorporation of a chlorine atom and the data is consistent with this being parent related. A metabolite scheme showing the final structures elucidated is shown in Fig. 5.

DISCUSSION There are several stages to the production of gas-phase charged ions in an electrospray source; these include the production of charged droplets, the reduction in droplet size through heat and coulombic eruptions and finally the release of ions into the gas phase from these very small highly charged droplets.[32] The production of charged droplets at the capillary is due to a high electric field produced at the tip of the emitter, which results in the formation of a Taylor cone where charged droplets are produced/ejected.[33] These droplets (in high flow electrospray) can then undergo significant desolvation of solvent molecules as they pass through a heated region within the source environment.[32] As these droplets reduce in size, the charge density within

the droplet increases and exceeds the Rayleigh limit (defined as the maximum amount of charge contained within a droplet). At this point, the repulsion of the ions of the same polarity overcome the surface tension of the droplet and cause coulombic fission where the droplet explodes/bursts producing smaller more stable droplets, by ejecting analyte.[34] Several advantages are produced by reducing the chromatographic flow rate prior to ionisation and sample detection within a mass spectrometer. These include improved sample sensitivity due to improved ionisation and transportation efficiency.[25] A smaller liquid volume results in a higher proportion of analyte ions relative to liquid molecules. In addition, when compared to high flow rate systems, a smaller solvent volume is transferred to the high vacuum region of the mass spectrometer.[21] These lower flow systems have been described as producing a ’micro electrospray ionisation’ and the ions produced by applying the high voltage to the emitter have a significantly higher analyte to solvent ratio per unit volume.[25] The droplets produced from the reduced flow rate in the nano tile are such that they are already very small, with fewer solvent molecules to remove no desolvation heater is required in this system. The smaller quantity of solvent molecules readily evaporates resulting in a high surface to volume ratio and a higher proportion of analyte at the surface.[33] These droplets continue to lose solvent molecules resulting in further ejection of analyte. The lower solvent volume and appropriate source geometry can therefore result in more of the analyte molecules being released into the gas phase (per unit volume) and the observed sampling efficiency being significantly greater than that of a standard higher flow electrospray source. Although separations in microfluidic devices offer a number of advantages, one major hurdle when working with small sample volumes is to gain sufficient detection sensitivity. The importance in the ability of the mass

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Figure 5. Midazolam (MDZ) metabolites detected in blood for the mouse strains severe combined immunodeficient (SCID) and Genotype: uPA+/+/SCID (PXB).

R. Gallagher et al. Table 4. Comparison of metabolites detected in chimeric mice from two mass spectrometry systems with (Xevo G2-S) and without (Xevo G2) the ’StepWave™’ ion optics. ✓indicates metabolite detected Metabolite ID and assignment Peak ID Parent M1 M2 M3 M4 M5 M6 M7 M8

StepWave™ Q-Tof

Q-Tof

Assignment

SCID mouse

PXB mouse

SCID mouse

PXB mouse

Midazolam (MDZ) 1’-Hydroxy-MDZ 4’-Hydroxy-MDZ 1’,4’-Dihydroxy-MDZ Dihydro-MDZ 1’-Hydroxy-O-glucuronide MDZ Dihydroxy-MDZ N-Glucuronide MDZ + H2O3

✓ ✓

✓ ✓ ✓

✓ ✓

✓ ✓ ✓ ✓

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spectrometer to efficiently extract and focus ions is highlighted when a comparison is made of the metabolites detected between two Q-tof mass spectrometer instruments that differ in the design of the ion source to mass analyser interface.[35,36] In this previous study, where the same microfluidic source interface was tested, only midazolam and three metabolites were reliably detected in the mouse compared to eight metabolites in the latter analysis on the improved interface design. Table 4 summaries the metabolites seen in the PXB and SCID mice on the two systems. The latter mass spectrometer is fitted with the ion optic device known as the StepWave™ ion guide. This is a stacked ring radiofrequency (RF) device that is differentially pumped such that it allows the transfer of ions from the source region to the mass analyser but also the removal of the neutral material. Increased background noise levels would be observed if the neutrals were allowed to enter the mass spectrometer. The system is designed to allow the capture of all of the expanding ion cloud from the source before focusing the ions off axis into the mass analyser.[31] The result of this is a marked improvement in sensitivity that, along with the ionisation efficiency improvements from the nano-flow source, allowed a greater number of metabolites to be detected and characterised here. In the present study the expected difference in the midazolam exposure between the control and chimeric animals was confirmed with an observed higher exposure of midazolam in the PXB mice. A 2.2-fold higher AUC0–3h value of the predominant primary metabolite, 1-hydroxymidazolam, was observed in the SCID mice compared to that in the PXB mice from data generated. This result cannot be compared to published data as an incomplete time profile was previously measured at this given dose level. However, the higher AUC value observed in samples of SCID mice compared to PXB mice are in line with what has been reported for the 0.1 and 10 mg/kg dose comparing 0–1 h and 0–8 h, respectively.[29] This data was produced from 5 μL blood volumes on more traditional analytical systems comprising of HPLC in conjunction with a triple quadrupole mass spectrometer. The accuracy and precision measured for the QCs on the microflow system were all bar one found to fall within the acceptance criteria of the study parameters. The relatively poor precision value (46%) measured for the 1’-hydroxy-O-glucuronide

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QC is believed to be a consequence of this standard degrading with time as the relative amount measured exhibited a decreased trend in each analytical run. Metabolite profiling of blood samples and quantification of the circulating metabolites has highlighted a marked difference in the formation of primary and secondary metabolites of midazolam in SCID and PXB mice. The ability of the PXB mice to generate circulating metabolites previously identified in human-derived (but not mouse) samples such as the 1’-hydroxymidazolam glucuronide and 4’-hydroxymidazolam shows the potential of this chimeric PXB mouse model to provide a first insight into human metabolism much earlier than in the drug discovery program. Compared to previous studies on these mouse strains this new analysis technique reveals the presence of four additional metabolites observed in the SCID mouse (M2, M4, M6 and M8) and two in the PXB mice (M7 and M8). Of the four additional metabolites detected in the SCID mouse all four have previously been observed in samples generated by other types/strains of mice where midazolam metabolism has been investigated.[30] The increase in sensitivity obtained from combining nanoUHPLC along with nano-electrospray is highlighted in the detection of a metabolite not previously reported in the literature, e.g. M8, which to our knowledge has not previously been reported. In this study a single injection of 18% of the plasma extract from single micro-litre blood time points was all that was required to obtain both full qualitative and quantitative metabolite profiles and allows for repeat analysis if required. The use of this highly sensitive microfluidic tile also helps the ethical treatment of animals by reducing both the number of animals needed for a study and the blood sampling volumes required.[37]

CONCLUSIONS The improvements in the enhanced ionisation efficiency from the prototype microfluidic device in conjunction with nanoUHPLC and mass spectrometry was sufficiently sensitive for the identification and quantification of midazolam metabolites from a single micro-litre of whole blood. The stability and sensitivity of the system allowed for the quantitation of all three previously identified human metabolites. The presence of all these metabolites in the

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Microfluidic device for identification and quantitation of metabolites SCID mice (at lower concentrations) had not been previously been observed. Additional metabolites known to occur in human metabolism were also qualitatively determined to be present in the mouse extracts for the first time. These included 1’,4’-dihydroxymidazolam and a midazolam diol. The results show that the PXB chimeric mouse is capable of forming a more human-like metabolite profile for midazolam compared to the SCID mouse and thus may provide a model system for predicting circulating human metabolites. The increase in sensitivity is also highlighted by the characterisation of a new metabolite not previously reported.

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Rapid Commun. Mass Spectrom. 2014, 28, 1293–1302

The application of a new microfluidic device for the simultaneous identification and quantitation of midazolam metabolites obtained from a single micro-litre of chimeric mice blood.

Improvements in the design of low-flow highly sensitive chromatographic ion source interfaces allow the detection and characterisation of drugs and me...
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