©2014 Waters Corporation MKT14082

イオンモビリティー質量分析計を用いたフルオロキノリン類の分子内における 複数のプロトン化部位とフラグメントパターンの違いの探索 Discovery of multiple sites of intra-molecular protonation and different fragmentation patterns within the fluoroquinolones using ion mobility mass spectrometry

フルオロキノン類は、（a）感染症の防止や（b）成長促進など様々の用途で、家畜に投与される抗菌薬の一種である。米国食品医薬品局は、薬剤耐性菌の人への拡散に関する懸念から、2005年9月1-2に、エンロフロキサシンとシプロフロキサシンの家畜生産における使用を禁止した。EUでは、2006年から3,4、抗菌性成長促進剤（AGPs）の畜産業における使用を禁止している。

数多くの異なるタイプの質量分析計が、日常的に動物用医薬品残留（VDR）試験に使用されており、そこには、シングル四重極、タンデム四重極、イオントラップ、そして、最近では飛行時間型などが含まれている6,7,8。タンデム四重極質量分析計は、感度と選択性の観点から、定量測定において幅広い分野で採用されている。VDR試験においては、最も適切なMRMトランジッションの選択が重要であり、その実施と妥当性評価は、2002/657/ECのガイドライン9に従う必要がある。

高精細質量分析(HDMS)は、高分解能質量分析計と、衝突断面積の測定とそれに基づく分離が可能な、効率の高いイオンモビリティーを組み合わせたシステムである。これにより、化合物は、大きさ、形状及び価数による分離が可能となる。さらに、 HDMSEは、プリカーサーイオンとフラグメントイオンを、一回の測定で取得が可能なHDMSによる測定モードである。

本研究では、HDMSが、フルオロキノン系抗菌剤の確実な同定メソッドの開発において、重要な手法となりうるかについて評価を行った。 HDMSによって、夾雑成分を多く含む豚の筋肉組織からの抽出液を測定し、フルオロキノン系抗菌剤の残留検出を行った。

Figure 1. A schematic representation of a SYNAPT G2-S HDMS and illustration of the mechanism of travelling wave ion mobility separations.

・本研究では、イオンモビリティーを利用し、フルオロキノン系抗菌薬を測定する際に観測される、 複数のプロトン化部位を網羅的に解析した。

・観測されたプロトマーについて、衝突断面積(CCS)値を算出し、同定に利用した。

・ HDMSE法を試験室間の測定の違いを比較する実用的な手法として評価した。

Sample Preparation

The extracts of porcine muscle tissue were kindly provided by RnAssays for the purposes of this study. Briefly, known blank porcine muscle was fortified with 25 different antimicrobial compounds (from the fluoroquinolone, tetracycline and macrolide classes) at the relevant to the EU MRL concentrations prior to extraction. The tissue samples were mechanically homogenised in the presence of an aqueous/organic extraction solvent followed by a centrifugation step. An aliquot of the supernatant was removed and placed in autosampler vial for subsequent LC-MS analysis.

MS Conditions

MS system: Waters SYNAPT® G2-S HDMS Ionization mode: ESI+ Acquisition Mode: HDMSE Mass range: m/z 50 – 1200 Acquisition Rate: 4 spectra/sec Capillary voltage: 2.0kV Cone Voltage: 25 V Source temp: 120 oC Desolvation temp: 550 oC Drift Gases: N2 MSE High energy: 15.0—45.0 V (ramped) IMS Wave Velocity: 900 m/s IMS Wave Height: 40V Resolution: 20000 (FWHM) UPLC Conditions

LC system: ACQUITY UPLC® System Column: ACQUITY BEH C18, 1.7 µm, 2.1 x 50 mm, @ 45 oC Mobile phase A: Water (0.1% formic acid) Mobile phase B: Acetonitrile (0.1% formic acid) Gradient Table: Injection Volume:10µL Gradient: Time (min) Flow Rate %A %B Initial 0.600 95.0 5.0 1.00 0.600 95.0 5.0 8.00 0.600 5.0 95.0 9.00 0.600 95.0 5.0

Data Acquisition and Processing

Data were acquired using MassLynx and processed using prototype UNIFI software. Fluoroquinolone standard mixtures were used to generate a scientific library containing retention times, fragment ion elemental compositions and CCS values for fluoroquinolone protomers. This permitted non-targeted data acquisition with a targeted screen to be performed using HDMSE data generated from porcine extracts.

For the assay performed MassLynx data was acquired and has been processed using prototype UNIFI software, allowing ion mobility data to be processed in a conventional workflow within new scientific information system for non-targeted accurate mass screening applications.

The antibiotic ciprofloxacin was determined to elute at retention time 2.19 minutes using the generic gradient conditions employed. This can be seen in Figure 2 where the base peak ion chromatogram is presented with the component plot summary for nine identified fluoroquinolone antibiotics. However, if the data is reviewed using the component drift plot summary, it can be seen that actually 18 fluoroquinolone species have been identified.

Each fluoroquinolone is comprised of two protomers i.e. protonation at two different sites on the molecule, which have been mobility separated, these are presented in Figure 3. These gas phase components, although they only differ with site of protonation, as can be seen for ciprofloxacin in Figure 4, have been separated by 0.5 milliseconds in this ion mobility study10.

The mobility separation obtained has enabled the individual precursor ion and fragments of all nine fluoroquinolones to be obtained in one analysis. From the single component MSE fragmentation spectra it was possible to determine that for ciprofloxacin the two mobility separated species resulted from protonation taking place on either the acidic and basic group.

Figure 2. UPLC HDMSE precursor base peak ion chromatogram for a solvent standard containing 25 antibiotic compounds (top) and the component plot summary view (bottom) for fluoroquinolone compounds detected between 2 and 2.6 minutes.

Figure 3. Component plot summary for the identified fluoroquinolone antibiotics.

From method development with standards, specific drift times and collision cross sections are generated simultaneously, providing further specific information to be entered into the scientific library, hence veterinary drug residues can be indentified based on retention time, accurate mass, fragments and CCS. For ciprofloxacin, estimated CCS values of 108.7 Ǻ2 and 119.1 Ǻ2 have been determined. For ciprofloxacin the fragments at m/z 314 and m/z 231 in Figure 5 are believed to form from a species where ionisation has taken place on the acidic group. Protonation occurring on the basic group results in the formation of fragment ions at m/z 288 and m/z 245 .

Once the CCS values and fragments of the individual fluoroquinolones were entered into the scientific data base, a series of porcine extracts were screened to determine the presence of fluoroquinolones. An example of the result of the IMS screening results obtained are shown in Figures 6 and 7, where the identification of two protomers of danofloxacin in porcine muscle extract is presented. Illustration of the benefits of ion mobility resolution are presented where the software functionality has been used to resolve the matrix background from the identified component danofloxacin. Also it can be seen that for both protomers, mass accuracy < 1ppm was obtained, % CCS error is within 2% of the expected CCS values, the observed retention time was 2.36 minutes and the individual protomer precursor ion/fragmentation spectra have been obtained.

Figure 4. Intensity vs drift time for the protomers of ciprofloxacin with the hypothesised respective sites of acid/basic group protonation highlighted and determined estimated CCS values.

Figure 5. Ciprofloxacin acid and basic group protomer MSE fragmentation spectra generated using an IMS screening workflow in UNIFI.

Figure 6. Illustration of identification of two protomers of danofloxacin indentified in porcine muscle extract, where no ion mobility resolution spectral clean up has been selected.

Figure 7. Illustration of the identification of two protomers of danofloxacin in spiked porcine muscle extract, where ion mobility resolution spectral clean up has been selected.

References

1. Final Decision of the Commissioner, Docket # 2000N-I 57 1, Withdrawal of the approval of the new animal drug application for enrofloxacin in poultry, Department of Health and Human Services, U.S. Food and Drug Administration.

2. FDA Center for Veterinary Medicine, June 2, 1997, FDA Order Prohibits Extralabel Use Of Fluoroquinolones And Glycopeptides, http://www.fda.gov/AnimalVeterinary/NewsEvents/CVMUpdates/ucm127904.htm

3. Official Journal of the European Union, L268:29-43, Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition.

4. Official Journal of the European Union, L24:1-8 Annex I, Council Regulation (EEC) No 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin

5. J. AOAC Inter.2005; 88:1179-1192.Verdon E., Couedor P., Roudaut B. and Sandérs P. 6. Rapid Commun. Mass Spectrom. 2009; 23: 985–998. A. Kaufmann, P. Butcher, K. Maden,

M. Widmer, K. Giles and D. Uria 7. Anal Bioanal Chem (2012) 403:2891–2908. H. G. J. Mol, P. Zomer & M. de Koning 8. J. Am. Soc. Mass Spectrom. (2012) 23:1569. T. R. Croley, K. D. White, J. H. Callahan, S. M.

Musser 9. Commission Decision (2002/657EEC) Official Journal of the European Communities 2002 10. J Mass Spectrom. (2012) 47(6):712-9. Lalli PM, Iglesias BA, Toma HE, de Sa GF, Daroda

RJ, Silva Filho JC, Szulejko JE, Araki K, Eberlin MN

・本研究で対象としたフルオロキノン類のイオン化の詳細な解析を行うことにより、UPLC/HDMSE

法が、メソッド開発において重要な役割を果たしうることが示された。 ・分子内のプロトン化部位が異なるイオン種の解析は、イオンモビリティー質量分析計によって行 われ、また、異なるイオン種のフラグメントイオンを測定することも可能であった。 ・すべての化合物において、それぞれのイオン種ごとのプリカーサー及びプロダクトイオンのスペクトル を、一回の測定で取得することができた。 ・ HDMSEが示す複数のプロトマーの存在は、試験室間において報告されることのあるMRMトラ ンジッションの挙動の相違を説明できる可能性が示唆された。 ・ノンターゲットのスクリーニング試験において、保持時間とプリカーサーイオン及びフラグメントイオン の精密質量に加えて、衝突断面積（CCS）も、化合物同定の基準として使用可能と考えられた。 ・イオンモビリティー分離は、効果的に対象化合物のピークと試料マトリックスによる夾雑成分を分 離することが可能であり、煩雑な試料前処理やクロマトグラム分離への依存度を減らすことが可 能であった。

Multiple protonated species have been observed for the fluoroquinolone antibiotics screened. The extent of the protonation multiplicity and is experimental variation is still being investigated. This data confirms that further consideration should be given to method development and the means of analysis chosen, since the ratio and formation of the protomers varies with eluent flow rate, capillary voltage, cone voltage and extract composition. If MRM is the method of choice, consideration of the experimental conditions used and the specific transitions selected is imperative. The data presented illustrate that consistency in MRM transitions in inter/intra laboratory studies could easily be misinterpreted within and between different laboratories and explain the challenges of achieving reproducible results for these types of compounds. Ion mobility is a valuable tool for both method development and analyte confirmation purposes.

OVERVIEW

INTRODUTION

RESULTS & DISCUSSION

METHODS

CONCLUSION

佐藤太1 ; Michael McCullagh2 ; Sara Stead2 ; David Eatough2 ; Kieran Neeson2 ; Jeff Goshawk2 1日本ウォーターズ株式会社 ; 2ウォーターズコーポレーション