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Keywords:

  • Chiral separation;
  • DART-MS;
  • Jasmonic acid;
  • 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol;
  • NPLC

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

Normal phase chiral LC (NPLC) has been proved to be powerful and efficient for chiral separation. However, the combination of NPLC with ESI or atmospheric pressure chemical ionization MS is restricted by the poor ionization efficiency and thermal fragmentations of analytes to some extent. Direct analysis in real time MS (DART-MS) is an ambient ionization technique that shows high ionization efficiency of the analytes in the normal phase mobile phase. In this work, we coupled chiral NPLC to DART-MS for the chiral qualitative and quantitative analysis of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and jasmonic acid enantiomers. Satisfactory results for the enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol operating in the positive mode were obtained in terms of linearity (2.5–250 μg/mL, R2, 0.999–1.000) and repeatability (25 μg/mL, RSDs, 4.7–5.6%). Moreover, chiral NPLC-DART-MS resulted in the simultaneous chiral separation and detection of jasmonic acid enantiomers, which are very difficult to be analyzed by NPLC-ESI-MS and NPLC-APCI-MS. Compared with the coupled techniques of NPLC-ESI-MS and NPLC-APCI-MS, NPLC-DART-MS showed advantages in increasing the ionization efficiency and reducing the in-source thermal fragmentation of analytes.

Abbreviations
APCI

atmospheric pressure chemical ionization

APPI

atmospheric pressure photoionization

DART

direct analysis in real time

EIC

extracted ion chromatogram

JA

jasmonic acid

NNAL

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

NPLC

normal phase liquid chromatography

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

Chiral separation plays an important role in life science, especially in drug discovery and development, because chiral drug enantiomers often exhibit remarkably different effects in pharmacological activity, transport mechanism, pathway of metabolism, and toxicity. Although great success has been achieved in asymmetric synthesis, enantioselective reactions are still restricted to the use of enantiomeric pure materials, such as chiral substrates, chiral auxiliary, and chiral catalysts [1, 2]. Hence, development of chiral separation methods has been a hot area in analytical and preparative chemistry. LC is still the most popular and highly acceptable technology in the field of chiral separation [3]. Compared with reversed-phase LC, normal phase LC (NPLC), using a polar stationary phase and nonpolar or weak polar solvents as mobile phase, could give more efficient chiral separations as it could provide stronger interaction between the chiral stationary phase and the analytes [2, 4, 5].

MS could increase the selectivity of detection and give structural information of analytes due to the possibility to record the MS or MSn spectra. Among the ionization techniques developed for LC-MS, ESI [6] is the most commonly used ionization technique, followed by atmospheric pressure chemical ionization (APCI) [7] or atmospheric pressure photoionization (APPI) [8]. All of them have their own advantages as well as drawbacks. Although ESI covers a wide range of molecule weight ranging from small molecules to large biomolecules, the use of compatible mobile phase in LC is necessary. NPLC, using the mobile phase containing large percentage of alkanes such as n-hexane [9-12] would lead to lower ionization efficiency of the analytes in the mobile phase. APCI shows less limitation toward nonprotonic mobile phase, but it is a less soft ionization technique than ESI due to thermal fragmentation [13]. It generates fragments relative to the parent ion and makes the mass spectrum more complex [13]. In addition, both ESI and APCI are generally considered to be incompatible with NPLC mobile phase due to the concern of potential explosion hazard caused by APCI corona needle discharge or ESI needle high voltage discharge [12]. APPI is an alternative ionization technique as there is no concern of potential explosion hazard. However, APPI needs the adding of dopant to increase the sensitivity of the target analytes in some cases, and its application is still limited at present. In a word, it is still necessary to develop a method combining the powerful chiral separation ability of NPLC with high sensitivity and selectivity of MS.

Direct analysis in real time (DART), introduced in 2005 by Cody and Laramee [14], is one of the most successful ambient ionization techniques. By using helium or nitrogen as the working gas, DART could produce heated metastable plasma in the glow discharge chamber and transfer the proton to analytes by the reactive species in the ambient atmosphere, such as water clusters [14, 15]. When coupled to various types of MS, DART-MS can provide a rapid and direct way to analyze different samples, such as direct analysis of activity assays from live samples [16-18], fast screening of target compounds in real samples [19-25], undamaged identification of ingredients in inks and alums [26-29], obtaining evidence of sexual assaults [30], reaction monitoring in drug discoveries [31], quantitative detection of warfare agents [32], and efficient analysis of environmental samples after stir bar absorptive extraction [33]. An additional advantage of DART is its relatively low tendency toward matrix effect, which has been confirmed by the direct analysis of biological sample with complex matrix without sample clean up or chromatographic separation [34]. Very recently, the coupling of LC to DART-MS has also been reported by Klampfl et al. [35, 36], showing that DART is compatible with the mobile phase containing nonvolatile phosphate buffers due to the indirect contact of the mobile phase with the ion source and the MS inlet [37, 38]. Based on the ionization mechanism and wide applications, we assumed DART could be used as a compatible ionization technique for NPLC-MS.

In this work, we coupled NPLC with DART-MS by a PEEK Tee splitter to combine the powerful chiral separation ability of NPLC and the convenient detection ability of DART-MS. Desolvatation and ionization of the effluent from NPLC occurred as soon as it arrived at the metastable helium flux produced by DART. This technique has been applied for the chiral separation and determination of enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and jasmonic acid (JA) under both gradient and isocratic elution. Compared with NPLC-ESI-MS and NPLC-APCI-MS, NPLC-DART-MS showed advantages in increasing the signal intensity and reducing in-source thermal fragmentation of analytes. In addition, the simple splitting setup allows simultaneous detection on DART-MS and sample collection for further characterization without influences from the heated helium plasma.

2 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

2.1 Chemicals and reagents

Racemic NNAL was purchased from Toronto Research Chemicals (North York, Ontario, Canada). Racemic JA and HPLC-grade ammonia solution (25% in water, NH3·H2O) were from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol, n-hexane, and isopropanol were all obtained from Dikma Technology (Richmond, VA, USA). Purified water was provided by Hangzhou Wahaha Group. (Hangzhou, Zhejiang, China).

Stock solutions of racemic NNAL and JA were prepared in methanol at 1.0 mg/mL and stored at –20°C until we needed to prepare other solutions with required concentrations, and then the remained stock solutions were stored at –20°C again. The experimental results demonstrated that the stock solutions were stable for more than 6 months.

2.2 NPLC-DART-MS system setup

All LC experiments were performed on an Agilent 1200 HPLC system (Agilent Technologies, Karlsruhe, Germany). The DART®-SVP ion source (IonSense, MA, USA) was coupled to an Agilent 6530 Accurate-Mass QTOF MS (Agilent Technologies, CA, USA) after removing the Agilent Jet Stream ESI source. The LC system was coupled to DART-MS through a simple interface shown in Fig. 1. After splitting by a PEEK Tee junction (Agilent Technologies), the mobile phase from LC was transferred to a fused-silica capillary with the polyimide coating removed (SinoSumtech, Hebei, China) through a PEEK junction (Agilent Technologies). The end of the fused-silica capillary was placed at the same height as the DART outlet and MS inlet. The distance between DART outlet and the end of the fused-silica capillary was 2 mm, and the distance between MS inlet and the end of the fused-silica capillary was 10 mm. The splitting effluent from the PEEK Tee junction was delivered to waste or collected for further study. Different from the work which used a Tee splitter to construct confined interface between the DART ion source outlet and mass spectrometer sampling orifice [39], the PEEK Tee junction was just used to adjust the splitting ratio in our experiment, and the ionization still proceeded in open-air.

image

Figure 1. Schematic of the setup used for the coupling of NPLC to DART-MS: (1) UV detector; (2) PEEK transfer tube from NPLC; (3) PEEK Tee junction; (4) collector for split effluent; (5) PEEK junction; (6) fused-silica capillary (100 μm id, d1 = 2 mm, d2 = 10 mm).

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2.3 LC conditions for chiral separation of racemic NNAL and JA

The chiral separation of NNAL was performed on a CHIRALCEL AS-3 (4.6 × 150 mm, 3 μm, Daicel, Japan) column under binary gradient elution with n-hexane/isopropanol (9:1, v/v, containing 0.1% NH3·H2O) as mobile phase A and isopropanol (containing 0.1% NH3·H2O) as mobile phase B. The gradient elution was set as follows: 0–15 min 8% B, 15.1–23.0 min 8–18% B, flow rate 1.0 mL/min. The chiral separation of JA was accomplished using 97:3 v/v n-hexane/isopropanol as the mobile phase on a CHIRALCEL OJ-H (4.6 × 250 mm, 5 μm, Daicel, Japan) column based on our previous work on LC-ESI-MS [40].

2.4 MS conditions

2.4.1 DART-MS

In the experiments, NNAL was detected under positive mode, and JA was detected on negative mode, and external standard method was used for the quantitation. The DART ion source was operated with helium for ionization mode and nitrogen for standby mode. After optimization, the DART operation parameters for positive and negative modes were, respectively, set as follows: discharge needle voltage 6000 V, gas temperature 350°C, gas flow rate 2 L/min, and grid electrode voltage 350/–350V. The QTOF MS was tuned and calibrated with Tuning Mix (Agilent P/N G1969–85000). The MS parameters for positive and negative modes were, respectively, set as follows: capillary voltage –1000 V/+1000 V, fragmentor 175/155V, and skimmer 65/65V. The MS data were acquired at a rate of 1.00 spectra/s in the mass range of m/z 100–600 by MassHunter Data Acquisition B.02.00 (Agilent Technologies) and were analyzed with MassHunterQualitative Analysis B.02.00 (Agilent Technologies).

2.4.2 ESI-MS and APCI-MS

ESI-MS experiments were performed on the same QTOF MS equipment as DART-MS. The optimized parameters of Agilent Jet Stream ESI source for positive mode were as follows: drying gas temperature, 325°C, drying gas flow rate 10 L/min, nebulizer gas pressure 50 psig, sheath gas temperature 325°C and capillary voltage –3500 V.

APCI-MS measurements were performed on an Agilent 6510 QTOF MS, and the following optimized parameters were employed for positive mode: drying gas temperature 325°C, drying gas flow rate 5 L/min, nebulizer gas pressure 30 psig, vaporizer temperature 300°C, capillary voltage –2500 V, and corona needle current 4000 nA.

2.5 Circular dichroism measurement of separated enantiomers

The single enantiomer fractions of NNAL and JA were manually collected and confirmed using circular dichroism. Their circular dichroism spectra were recorded on a JASCO-J810 spectrometer (Jasco, Japan). The light path length of the quartz cells used was 1.0 cm.

3 Results and discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

3.1 Optimization of NPLC-DART-MS system

To confirm the capability of this setup (Fig. 1), one had to find out the suitable operation conditions to volatilize the normal phase and ionize the enantiomers when the effluent arrived at the metastable helium flux. Residual effluent at the end of the capillary would form liquid drop that could reduce the resolution. Therefore, the operation conditions of DART including helium flow rate and temperature, the position of the capillary end and the flow rate of the effluent were optimized. First of all, a PEEK Tee junction was designed for adjusting the flow rate of the effluent by splitting. The other parameters mentioned above were optimized with the change of the split ratio. Higher flow rate of the effluent could give high signal intensity of analytes on DART-MS, but need higher temperature and helium flow rate as well as smaller distance d1 (see Fig.1). The experiments showed that at the split flow of 12:1, the effluent from the capillary could be ionized fast and efficiently by DART without liquid residual and with acceptable MS response. In addition, the effluent out of the splitter could be collected conveniently for further characterization without being influenced by the heated helium from DART.

3.2 Chiral separation and quantization of NNAL

NNAL, one of the most important pulmonary carcinogens in tobacco products and cigarette smoke, was used to test the feasibility of the established system. Due to the chiral center at carbinol and the different orientations of the –N = O bond, NNAL has four enantiomers including (E)/(Z)-(+)-NNAL and (E)/(Z)-(–)-NNAL (Fig. 2A), showing remarkable differences in metabolism, pharmacokinetics, and carcinogenic activities [41, 42]. Figure 2B shows the typical NPLC chromatogram of NNAL on the chiral column with DAD. The NPLC was then coupled to DART-MS directly or after splitting with the PEEK Tee junction. Figure 2C and D showed the extracted ion chromatograms (EICs) of NNAL enantiomers obtained on DART-MS. When the mobile phase of NPLC at the flow rate of 1 mL/min was directly transferred to DART-MS without splitting, the signals obtained on DART-MS had an obvious peak broadening and the resolutions were decreased (Fig. 2D) compared to the chromatogram obtained on DAD (Fig. 2B). In order to reduce the band broadening, we added a PEEK TEE junction between NPLC and DART-MS to split the mobile phase from NPLC with the splitting ratio of 12:1, obtaining improved results (Fig. 2C), which were in consistent with the chromatograms obtained on DAD (Fig. 2B). Moreover, the intensity obtained after splitting was almost as high as that without splitting, although the sample amount was only 1/13 of the total volume. This is because when the NPLC was operated at 1.0 mL/min without splitting to form the liquid jet, the effluent from the NPLC just stayed on the DART outlet for a short time, and most of the effluent was flowed away rather than ionized. If the effluent from LC was split before reaching the DART outlet, the flow rate decreased and the effluent could stay for a longer time to be sufficiently ionized. In addition, the effluent at 1.0 mL/min without splitting easily resulted in residual in air, leading to a worse resolution. An additional advantage of this splitting setup was allowing convenient sample collection for further characterization without being influenced by the heated helium plasma. In our experiments, each collected effluent out of splitter was tested by using circular dichroism to confirm the identification of each peak obtained by NPLC-DART-MS (data not showed). In a word, both the improved resolutions and acceptable peak intensities demonstrated that splitting setup was suitable for the coupling of NPLC to DART-MS.

image

Figure 2. (A) Structures of interconvertible geometric isomers of NNAL with the (E)-isomer and (Z)-isomer; (B) chromatogram of NNAL analyzed by NPLC with UV detector; (C) extracted ion chromatograms (EICs) of NNAL obtained on NPLC-DART-MS after splitting; (D) EICs of NNAL obtained on NPLC-DART-MS without splitting . (* represent the chiral center; 1 is (Z)-(–)-NNAL; 2 is (E)-(–)-NNAL; 3 is (Z)-(+)-NNAL and 4 is (E)-(+)-NNAL.)

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Because the (E)-NNAL and (Z)-NNAL are interconvertible [43, 44], we did not discuss the quantitation of them individually. In the following study, (–)-NNAL was considered to be the total amount of (E)-(–)-NNAL and (Z)-(–)-NNAL, and (+)-NNAL to be the total amount of (E)-(+)-NNAL and (Z)-(+)-NNAL. The linearity of this method was evaluated from 2.5 to 250 μg/mL for both (–)-NNAL and (+)-NNAL. The calibration curve was obtained by plotting logarithms of the peak area to the logarithms of sample concentration with acceptable linearity, as listed in Table 1. The LOD at S/N = 3 were 0.5 μg/mL for both (–)-NNAL and (+)-NNAL, with RSDs of 4.7 and 5.6% at the concentration of 25 μg/mL (n = 6) and 4.4 and 4.2% at 2.5 μg/mL (n = 6).

Table 1. Method validation for the chiral separation of NNAL using NPLC-DART-MS, NPLC-ESI-MS, and NPLC-APCI-MS
  Regression equationaR2Linear range (μg/mL)LOD (μg/mL)LOQ (μg/mL)RSDb (n = 6)
  1. a

    x and y mean log[concentrations of determinants] and log[peak areas], respectively.

  2. b

    The concentration is 25 μg/mL.

  3. c

    The method was invalid for quantitative work.

DART(−)-NNALy = 0.897x + 4.8251.0002.5–2500.52.54.7%
 (+)-NNALy = 0.894x + 4.8810.9992.5–2500.52.55.6%
ESI(−)-NNALc
 (+)-NNAL
APCI(−)-NNALy = 1.180x + 5.2130.9952.5–2500.52.51.3%
 (+)-NNALy = 1.172x + 5.2300.9962.5–2500.52.51.7%

3.3 Comparisons with ESI-MS and APCI-MS

For comparison of DART-MS with other ionization techniques, the NPLC system using high percentage of n-hexane for the chiral separation of NNAL was coupled to ESI-MS and APCI-MS for further detection.

From the chromatogram obtained on DAD (Fig. 2B) and the EICs obtained on DART-MS (Fig. 2C), we can see that the relative intensity of NNAL enantiomers agreed well with each other in the full range of elution. The ratio of the peak areas for the four isomers was 1:4:1:4 on DAD chromatogram and EICs obtained on DART-MS. This indicated that DART showed high ionization efficiency toward the analytes regardless of the content of the nonprotonic n-hexane in the mobile phase. However, when the same NPLC system was coupled with ESI-MS, the ratio of the peak areas for the four isomers was 1:1:1:5 (Fig. 3B), which could not represent the real relative content of the four isomers. In the first 15 min, the mobile phase contained 82.8% of n-hexane resulted in lower peak intensities for isomer 1 and 2; while in the next 8 min, the percentage of n-hexane decreased linearly from 82.8 to 73.8%, the peak intensity for isomer 4 was about five folds to that of isomer 2. These results demonstrated that ESI was not appropriate for NPLC because of the lower ionization efficiency toward the analytes in the mobile phase with high percent nonprotonic n-hexane.

image

Figure 3. (A) EICs of NNAL obtained on NPLC-DART-MS after splitting; (B) EICs of NNAL obtained on NPLC-ESI-MS with the same splitting; (C) mass spectrum of NNAL obtained on APCI-MS; (D) mass spectrum of NNAL obtained on DART-MS. (1 is (Z)-(–)-NNAL; 2 is (E)-(–)-NNAL; 3 is (Z)-(+)-NNAL and 4 is (E)-(+)-NNAL.)

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The same NPLC system was coupled to APCI-MS for further study. APCI was chosen for further study because of its more similar ionization mechanism to DART than that of ESI [45]. Similar to DART-MS, APCI-MS had the linear range of 2.5–250 μg/mL with the LODs of 0.5 μg/mL for both (–)-NNAL and (+)-NNAL, as listed in Table 1. However, there were other fragment ions besides the molecule ion (Fig. 3C) on the mass spectrum due to the thermal fragmentation. Compared with the mass spectrum obtained on DART-MS that showed only the molecular ion, the fragmentations by APCI-MS made the mass spectrum more complex, especially for the sample with various components. Hence, both of the comparisons demonstrated that DART-MS was a more compatible detector for chiral NPLC than ESI-MS and APCI-MS.

3.4 Chiral separation and detection of JA

This work aimed at evaluating whether DART-MS could be regarded as a suitable detector for NPLC even in the presence of high percentage of nonprotonic mobile phase. Thus, the chiral separation of JA by chiral NPLC using 97% n-hexane and 3% isopropanol as mobile phase was further tested on our setup.

As an essential plant hormone involved in plant growing and defense system, JA has two chiral centers at C-3 and C-7, resulting in four possible stereoisomeric forms including (3R,7R)-(-)-JA, (3S,7S)-(+)-JA, (3S,7R)-(-)-JA, and (3R,7S)-(+)-JA (Fig. 4A). Commercially available JA is a racemic mixture of the four isomers. Although chiral reversed-phase LC using a mixed mobile phase of ACN and 0.1% triethylammonium acetate in water (30:70, v/v; pH 4.0) showed the ability to separate the four enantiomers into two parts and distinguishing the (-)-JA and (+)-JA [46], it was unsuitable for plant sample analysis since the naturally existing isomers are only (3R,7R)-(-)-JA and (3R,7S)-(+)-JA [47]. Recently, the complete separation of the four enantiomers was obtained by CE [48] and NPLC using n-hexane/isopropanol (v/v, 97:3) as mobile phase in our lab [40]. However, no signal could be obtained for these isomers of JA on ESI-MS without a postcolumn addition of isopropanol with 0.1% NH3·H2O as the make-up solvent.

image

Figure 4. (A) Structures of the four JA stereoisomers; (B) chromatogram of JA analyzed by NPLC with UV detector; (C) EICs of JA obtained by NPLC-DART-MS after splitting. (1 is (3S, 7R)-(–)-JA; 2 is (3R, 7R)-(–)-JA; 3 is (3R, 7S)-(+)-JA; 4 is (3S, 7S)-(+)-JA.)

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In this work, the NPLC was directly coupled to DART-MS without any make-up solvent. Figure 4C illustrates the EICs obtained by DART-MS for the four JA enantiomers, showing baseline separation and detection with high ionization efficiency. Calibration curves constructed in the range between 1.0 and 200 μg/mL showed good linearity (R2 = 0.997) with an excellent RSD of 1.4% (n = 6, 20 μg/mL) and satisfactory LOD (0.5 μg/mL). This successful application further demonstrated the compatibility of DART-MS with NPLC. It is worth mentioning that this chiral NPLC system was also coupled to both ESI-MS and APCI-MS for JA detection, but no signal was obtained due to the bad ionization efficiency toward the analytes in high percentage of n-hexane.

4 Concluding remarks

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

NPLC-DART-MS, combining the powerful chiral separation capability of NPLC and the qualitative detection ability of DART-MS, has been developed in this work. Compared to the conventional ionization techniques (APCI and ESI), DART showed higher ionization efficiency and less thermal fragmentation in tolerance with the nonprotonic solvent in the mobile phase. Using the simple splitting setup, fast and effective chiral analytical methods for NNAL and JA enantiomers were established under gradient and isocratic elution, respectively. Satisfactory results for NNAL and JA were obtained in terms of linearity, sensitivity, and repeatability. Besides, the splitting setup allows convenient sample collection for further characterization without influences from the heated helium plasma. In a word, this setup could contribute to the development of convenient and applicable chiral separation and detection methods based on NPLC-MS.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References

This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 21027012 and 90717002) and the Fundamental Research Funds for the Central Universities.

The authors have declared no conflict of interest.

5 References

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials and methods
  5. 3 Results and discussion
  6. 4 Concluding remarks
  7. Acknowledgments
  8. 5 References