Rapid analysis of benzalkonium chloride using paper spray mass spectrometry

Journal of Pharmaceutical and Biomedical Analysis 145 (2017) 151–157

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Rapid analysis of benzalkonium chloride using paper spray mass spectrometry Jingjing Liu a,b , Wenjie Deng a , Muqian Yu a , Ruizhi Wen a , Shouzhuo Yao a , Bo Chen a,∗ a Key Laboratory of Phytochemical R&D of Hunan Province and Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, Hunan Normal University, Changsha 410081, China b College of Chemistry and Chemical Engineering, University of South China, Hengyang, 421001, China

a r t i c l e

i n f o

Article history: Received 13 April 2017 Received in revised form 15 June 2017 Accepted 15 June 2017 Available online 20 June 2017 Keywords: Benzalkonium chloride Cation surfactant Biocides Paper spray mass spectrometry High performance liquid chromatography

a b s t r a c t A paper spray mass spectrometry (PS-MS) method for rapid and reliable analysis of benzalkonium chloride (BAC) in compound eye drops and body surface disinfectant was developed. The sample was dropped onto triangular filter paper, and high voltage (3.5 kV) was applied to form an electrospray. This method can provide the composition of benzalkonium chloride in samples without pretreatment, solvent or chromatographic separation, and the analysis time is only 10 s. The primary homologues C12 -BAC, C14 -BAC and C16 -BAC of benzalkonium chloride were quantitatively analyzed using PS-MS. Samples were subjected to simple dilution and quantified using the internal standard method. Ion trap mass spectrometry was scanned using SIM mode. The linear ranges of C12 -BAC, C14 -BAC and C16 -BAC were 1–100 ␮g mL−1 ; the linear regression coefficients were 0.998–0.999; the detection limits (LODs) were 0.1 ␮g mL−1 ; the limit of quantifications (LOQs) was <1 ␮g mL−1 , and the method validation indicated that the method precision and accuracy were good. Compared with HPLC-UV methods, there was no significant difference in the quantitative determination of the actual samples, but the analysis time for PS-MS is shorter (2 min). In addition, reagent consumption in PS-MS is small, and no chromatographic separation is needed, suggesting that PS-MS is especially suitable for high-throughput analysis. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Biocides are mass-produced chemicals that inhibit harmful microorganisms in many household and industrial applications. Benzalkonium chloride (BAC) is a class of quaternary ammonium compound (QAC) that is widely used as a cationic surfactant, with both hydrophobic and hydrophilic natures [1–4]. It is commonly added as a biocide in personal care products (eye drops, external cleaning solution), cosmetics and skin disinfection products. BAC is classified as a “Category III antiseptic active constituent” by the United States Food and Drug Administration [1]. When available data are insufficient to classify as safe and effective, and further testing is required, the ingredients should be classed as “Category III antiseptic active constituent”. It is generally accepted that BAC toxicity is low, but its improper use has serious consequences. For example, BAC was used in eye drops as a biocide from 1950. However, the BAC is not selectively toxic to bacteria, and there is increasing evidence that long-term use of this agent can cause or

∗ Corresponding author. E-mail address: [email protected] (B. Chen). http://dx.doi.org/10.1016/j.jpba.2017.06.030 0731-7085/© 2017 Elsevier B.V. All rights reserved.

exacerbate ocular surface disease [5,6]. The Ministry of Health of the People’s Republic of China stipulates the content of BAC in disinfectant products with various uses. The highest quality content of skin and mucous disinfectant is not more than 2 mg mL−1 . The limits of BAC content in different types of products are clearly defined in the Chinese cosmetics health standards and the cosmetics regulations of the European Union (Council Directive 76/768/EEC). BAC is a mixture of alkylbenzyldimethylammonium chlorides [C6 H5 CH2 N(CH3 )2 RCl], in which C12 , C14 , C16 -BAC are the main compounds (Fig. 1). Because their antibacterial effects come from the structure of alkyl chains and quaternary ammonium groups, the difference in alkyl chain length results in different antibacterial capacities and irritation [7,8]. The three homologues have different antimicrobial activity: C12 -BAC inhibits yeast and fungi, and C14 -, C16 -BAC inhibit gram-positive or negative bacteria. Therefore, benzalkonium chloride products are often used in a compound form to improve the scope of application of the disinfectant. The United States Pharmacopoeia (USP 34) indicates that the C12 -BAC content in the BAC formula is not less than 40%, C14 -BAC content is not less than 20%, and the combined amount of the two is not less than 70%. To date, many analysis methods for BAC have been reported, such as chemical titration [9], ultraviolet spectrophotometry [10],

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2. Materials and methods 2.1. Chemicals and reagents

Fig. 1. Structures of quaternary ammonium compounds.

Benzyldimethyldodecylammonium chloride (98%), benzyldimethyltetradecylammonium chloride (99%), benzyldimethylhexadecylammonium chloride (98%), sodium acetate (99%) and chromatography paper were purchased from J & K Scientific Ltd. (Beijing, China). Deuterated internal standards were prepared by our lab (The preparation processes of D2 C12 -BAC, D2 C14 -BAC and D2 C16 -BAC are shown in S.I.). Methanol and acetic acid were chromatography grade, and other organic solvents were analytical grade and purchased from Tianjin Chemical Reagent Manufacturing Co. (Tianjin, China). Water was purified using a Milli-Q purification system (Millipore Corp., Bedford, MA, USA). 2.2. Instruments and methods

capillary electrophoresis [11], differential spectrophotometry [12], immunoassay [13], liquid chromatography [1,14,15] and liquid chromatography-mass spectrometry [4,16]. Chemical titration, ultraviolet spectrophotometry and differential spectrophotometry can only determine total amounts, and cannot obtain the composition of the major homologous compounds of BAC. Immunoassays can only measure one component. HPLC and HPLC–MS can separate and determine many of these components. However, benzalkonium chloride is a cationic surfactant, and ion pair reagents must be added to the mobile phase. The ion pair reagent often affects the column life and is not conducive to the ionization of mass spectrometry due to its nonvolatility. Therefore, the development of new, highly selective analysis methods is particularly important. Paper spray mass spectrometry (PS-MS) is a well-developed ambient mass spectrometry (AMS) method [17,18]. It has been used for the fast, qualitative and quantitative analysis of complex mixtures such as blood, urine, saliva, tissues, cell culture, water and bacteria [19–27]. In terms of mechanism, paper spray ionization belongs to electrospray ionization (ESI) processes. It is very simple, i.e., the sample solution was ejected and ionized when a high voltage was applied to a paper triangle. Many analytes could be determined without preparation or less direct preparation. In our paper, a new and reliable method for the rapid qualitative and quantitative analysis of BACs in compound eye drops and body surface disinfectants was developed using PS-MS. The merits of this method are as follows: 1) the preparation is simpler than other reported methods; 2) the method has low solvent cost and saves labor; 3) the method is highly sensitive. A single PS-MS analysis can be completed in 2 min. Using the deuterium compound as an internal standard, this method can accurately determine the composition and content of BAC. Compared with the HPLC-UV method, there was no significant difference between the two methods in the quantitative determination of actual samples.

Paper spray was performed using an LCQ Advantage Max spectrometer (Finnigan Corp., Silicon Valley, CA, USA) using the following instrumental parameters. Capillary voltage, tube lens offset and capillary temperature were set at 10 V, 0 V and 200 ◦ C, respectively. Helium was used as a collision gas (no other pneumatic assistance, such as sheath or auxiliary gas was used), and the positive ion mode was employed. Paper triangles were cut from chromatography paper using a CUTOK DC craft cutting plotter (Hefei CNC Equipment Co. Hefei, China). The paper triangles had an angle of 38◦ and an area of ∼60 mm2 (base width = 9 mm, height = 13.2 mm). An XYZR (three-dimensional and angle regulator) moving platform (Figure S-1, Supporting Information) was constructed in-house and utilized to accurately locate the spray paper triangle tip with the MS cone. The paper triangle was held approximately 5–10 mm from the MS inlet. After directly adding 15 ␮L of the spray solution to the substrate, high voltage (+3.5 kV) was applied to the paper using an alligator clamp to generate electrospray, and mass spectra were recorded in SCAN or SIM mode. HPLC-UV was accomplished using an LC-20A high-performance liquid chromatograph (Shimadzu, Japanese) coupled with a UV detection and LC solution workstation and a Lichrospher CN column (4.6 × 300 mm, 5 ␮m, Hanbon Corp., Jiangsu, China) applied with the following instrumental parameters. Column temperature, flow rate, detection wavelength and injection volume were 35 ◦ C, 1.0 mL min−1 , 254 nm and 20 ␮L. The ratio of mobile A (0.1 mol L−1 sodium acetate solution, pH = 1) to mobile B (acetonitrile) was 48:52. 2.3. Preparation of solution and sample The stock solutions of C12 -BAC, C14 -BAC and C16 -BAC were prepared by dissolving each compound in methanol at a concentration of 3000 mg L−1 . A mixed internal standards solution of D2 C12 BAC, D2 C14 -BAC and D2 C16 -BAC was prepared in methanol at

Table 1 Names and abbreviations of nine samples purchased in local pharmacies. Sample

Name

Abbreviation

Eye drops-1 Eye drops-2 Eye drops-3 Eye drops-4 Eye drops-5 Eye drops-6 Eye drops-7 Sanitizer-1 Sanitizer-2

Compound Taurine Eye drops Compound Aspartate, Vitamin B6 , and Dipotassium Glycyrrhetate Eye drops Compound Allantoin Vitamin B6 -E and Aminoethylsulfonic Acid Eye Drops Bendazac Lysine Eye drops Naphazoline Hydrochloride, Chlorphenamine Maleate and Vitamin B12 Eye drops Brinzolamide Eye drops Naphazoline Hydrochloride, Chlorphenamine Maleate and Vitamin B12 Eye drops Hand-foot sterilization spray Sterilization spray

CT-ED CAVDC-ED CAVAA-ED BL-ED NHCMV-ED B-ED NHCMV-ED (without BAC) HS-S S-S

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Fig. 2. Optimization of PS-MS conditions for C12 -BAC in positive ion mode: (a) spray voltage; (b) capillary voltage; (C) capillary temperature. Error bars represent the standard deviation of three replicate measurements.

20 mg L−1 (each). All solutions were stored at 4 ◦ C. Working solutions were obtained by dilution at 1, 5, 25, 50 and 100 mg L−1 , and the addition of a 20 mg L−1 mixed solution of internal standards was performed at 1: 1 (V: V) for PS-MS analysis. Practical samples were purchased in local pharmacies (Changsha, China) (Table 1). For quantitative analysis of BACs using PS-MS, clear samples of eye drops were diluted 5-fold with methanol. The suspended eye drops (Eye drops-6) were centrifuged, and the supernatant was diluted 5-fold with methanol. Santizer-1 and santizer-2 were diluted 10- and 50-fold with methanol due to the higher concentration of BACs than eye drops, respectively. And the addition of a 20 mg L−1 mixed solution of internal standards was performed at 1: 1 (V: V) in diluted samples.

when the spray voltage was 3.5 kV, the intensity is not at the highest value, but the S/N is best, and thus 3.5 kV was used. According to Fig. 1(b) and (c), capillary voltage at 10 V and capillary temperature at 200 ◦ C were selected for PS-MS analysis of BACs. The spray solvent was investigated to improve the MS response of BACs. C12 -BAC was analyzed using methanol, methanol with 0.5 vol.% formic acid or methanol with 0.5 vol.% ammonia. The results indicated that BACs sprayed in acidic conditions had a lower response than for spray solvents, and there was no obvious difference between methanol and methanol with 0.5 vol.% ammonia, so methanol without additive was chosen.

2.4. Method validation

Fig. 3 shows the mass spectra of the actual sample directly subjected to PS-MS analysis (without pretreatment). Fig. 3a and b are the spectra of NHCMV-EDs; BAC were detected (mainly C12 -BAC) in Fig. 3b, while BAC cannot be detected in Fig. 3a, and the results are consistent with the actual situation. BAC homologues were detected in eight of the nine samples, and their compositions differed. Samples in Fig. 3b, d and e are domestic eye drops; their BAC formulas are mainly composed of C12 -BAC. Samples in Fig. 3c, f and g are imported eye drops; their BAC formulas are mainly composed of C12 -BAC, C14 -BAC and C16 -BAC. Fig. 3h and i are the mass spectra of the sanitizer spray. In Fig. 3h, the HS–S sample specification does not inform its disinfectant composition, but the mass spectrum shows that its main components were C12 -BAC, C14 -BAC and C16 -BAC. The mass spectrum in Fig. 3i shows that the main ingredients of the S–S sample were C12 -BAC, C14 -BAC and C16 -BAC, and a compound at m/z 388 which could be inferred to be C18 -BAC. To confirm the qualitative analysis of C12 -BAC, C14 -BAC, C16 BAC and C18 -BAC and investigate the selectivity of this method, the MS/MS function of LCQ was used to obtain the secondary cleavage mass spectra of m/z at 304, 332, 360 and 388 (Fig. 4). Bonds in the BAC homologues were broken, and mainly led to the removal of benzyl ions (m/z 304 → m/z 212, m/z 332 → m/z 240, m/z 360 → m/z 268, m/z 388 → m/z 296) and rearranged benzyl dehydrogenation ions (m/z 91). Peaks at m/z 212, 240, 268 and 296 correspond to [MC12-BAC CH3 C6 H5 Cl]+ , [MC14-BAC CH3 C6 H5 Cl]+ ,

Calibration standards were prepared using five standard samples, as described in Section 2.3, and calibration curves were generated by plotting the ratio of the analyte signal intensity to that of the IS versus concentration. The LOD and LOQ for each analyte were determined as the lowest concentrations that yield a signal to noise (S/N) ratio of 3.00 and 10.00, respectively. Recoveries and precisions of these analytes were evaluated by analyzing spiked samples at three spiked concentrations three times in a day (within-day) and on three separate days (between-day). 3. Results and discussion 3.1. Optimization of PS-MS condition for BACs To obtain suitable conditions for analysis of BACs, the spray voltage, capillary voltage and capillary temperature, which are important factors for ionization, were optimized. Spray voltage affects the ionization efficiency of PS-MS, and the capillary voltage and capillary temperature affect the amount of ions entering the mass spectrum. C12 -BAC (5.4 ␮g mL−1 ) was chosen as the model to investigate the PS-MS conditions and measured three times under various conditions. The results are shown in Fig. 2. Spray voltage is important for signal intensity and sensitivity. As shown in Fig. 1(a),

3.2. Method selectivity

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Fig. 3. Spectra of BACs in samples by PS-MS: (a) NHCMV-ED (without BAC); (b) NHCMV-ED; (c) CT-ED; (d) CAVAA-ED; (e) BL-ED; (f) CAVDC-ED; (g) B-ED; (h) HS-S; (i) S-S. Peaks at m/z 304, 332 and 360 correspond to [M–Cl]+ of C12 –BAC, C14 –BAC and C16 –BAC, respectively.

3.3. Quantitative analysis of BAC homologues

The calibration curves ranged from 1 to 100 ␮g mL−1 for C12 BAC, C14 -BAC and C16 -BAC and were plotted as concentration versus signal ratio of the analyte and the I.S. (Fig. 5). The linearity (R2 ) for all BAC homologues is >0.998. The LODs are 0.1 ␮g mL−1 , and LOQs are > 1 ␮g mL−1 (Table 2). The recoveries and percisions of eye drops-1 and sanitizer-1 are given in Table 3. The above data show that the PS-MS method is a high-throughput, high precision, accurate method.

3.3.1. Method validation In the quantitative analysis, the SIM mode was applied to simultaneously monitor benzalkonium ion responses of the target analytes and internal standards (m/z at 304, 306, 332, 334, 360, and 362). The standard solutions (prepared in Section 2.3) were analyzed by PS-MS, and each standard solution was injected five times. The calibration curves of C12 -BAC, C14 -BAC and C16 -BAC are given in Fig. 5, and the results of the method validation are shown in Table 2 and 3.

3.3.2. Application The developed method was applied to analyze the nine samples (Table 1). The indications for eye drops are relieving eye fatigue, eliminating inflammation and treating glaucoma and cataracts, among other uses. Specific information is described below. Origin: 4 from the inland, 3 from abroad. Suitable for ages: 1 for child, 6 for adult. Dosage: 6 liquids, 1 suspensions. Six of the above eye drops are added BAC. One sanitizer is not marked with the main ingredi-

[MC16-BAC CH3 C6 H5 Cl]+ and [MC18-BAC CH3 C6 H5 Cl]+ , respectively (Figure S-2, Supporting Information). The results are consistent with standards and literatures [4,28], demonstrating the high selectivity of the method.

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Fig. 4. MS/MS spectra of ions at m/z 304(a), 332(b), 360(c), 388(d).

Fig. 5. Calibration curves for BACs analyzed by PS-MS: (a) C12 -BAC, (b) C14 -BAC and (c) C16 -BAC. Error bars represent the standard deviation of five replicate measurements. Table 2 Analytical parameters of three BAC homologues by PS-MS. Compound

Working ranges (␮g mL−1 )

Regression equation

Correlation coefficients

LOD (␮g mL−1 )

LOQ (␮g mL−1 )

RSD (%, n = 5)

C12 -BAC C14 -BAC C16 -BAC

1–100 1–100 1–100

y = 0.066x + 0.022 y = 0.050x + 0.043 y = 0.046x + 0.013

0.998 0.998 0.999

0.1 0.1 0.1

<1 <1 <1

1–3.9 0.7–5.0 0.7–4.4

ent, and the other main ingredient is labeled BAC. The results are shown in Table 4. 3.3.3. Compared with HPLC-UV To investigate the accuracy of the PS-MS method, the BAC analytical method was established by HPLC-UV, and the above actual samples were analyzed. According to the United States Pharmacopoeia, an HPLC-UV method has been established using a cyanide columfn, and the specific conditions are described in 2.2. Because the HPLC-UV method has a higher detection limit, the standard solution with low concentration in the original working curves cannot be measured, and the reconstituted working curves of the three BAC homologues for HPLC-UV all ranged from 10, 50, 100, 150 and 200 ␮g mL−1 . After filtration, the eye drops without dilution were analyzed directly; suspended eye drops were centrifuged at high speed, and the supernatant was taken for analysis after filtration. Sanitizer-1 and sanitizer-2 were diluted 3-fold and 10-fold, respec-

tively, and treated as eye drops. The experimental results are shown in Table 5. As shown in Table 5 and 6, the results obtained by PS-MS are similar to those in the HPLC-UV assay. However, even when the undiluted eye drops were analyzed, the HPLC-UV method was unable to detect C16 -BAC in eye drops-1. This indicated that the LOQ of PS-MS is lower than the HPLC-UV method. A PS-MS mass spectrometry method was used to establish a calibration standard curve for a short time, while the HPLC-UV method takes longer; a single sample analysis using PS-MS takes 2 min, while HPLC requires 25 min. PS-MS does not require mobile phase and ion pair reagents. 4. Conclusions In this paper, a rapid method for the rapid analysis of BAC in compound medicines (i.e., eye drops and sanitizers) was established using PS-MS. After optimizing experimental conditions, the

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Table 3 Recoveries and precisions for three BAC homologues by PS-MS. Spiked sample

Compound

Eye drops-1

Sanitizer-1

Background concentration (␮g mL−1 )

Spiked concentration (␮g mL−1 )

C12 -BAC

34.9

C14 -BAC

12.2

C16 -BAC

5.3

C12 -BAC

233.5

C14 -BAC

242.0

C16 -BAC

56.0

10.0 20.0 40.0 10.0 20.0 40.0 10.0 20.0 40.0 100.0 200.0 300.0 100.0 200.0 300.0 100.0 200.0 300.0

Within-day (n = 3)

Between-day (n = 3)

Recovery (%)

RSD (%)

Recovery (%)

RSD (%)

103.1 106.6 98.5 104.1 102.5 104.4 96.1 98.4 95.3 99.7 106.9 98.5 100.3 97.6 102.7 96.0 105.7 98.3

3.29 1.92 1.36 2.35 2.64 2.85 3.31 1.45 3.71 1.02 3.22 2.22 0.66 5.27 4.73 0.43 2.73 1.36

102.3 103.3 99.1 103.2 102.9 101.3 97.5 99.1 94.4 98.9 103.3 97.9 100.1 99.6 100.5 96.0 105.7 98.3

2.33 3.12 1.99 2.87 3.98 1.23 4.19 3.22 4.21 2.65 2.98 3.86 1.78 4.90 5.53 1.23 2.77 2.02

Table 4 Measured concentrations for BAC homologues in samples using PS-MS. Sample

Experimental concentration

Eye drops-1 Eye drops-2 Eye drops-3 Eye drops-4 Eye drops-5 Eye drops-6 Eye drops-7 Sanitizer-1 Sanitizer-2

C12 -BAC

C14 -BAC

C16 -BAC

34.9 82.47 108.5 130.1 161.2 80.8 – 233.5 761.3

12.2 32.9 – – – 18.6 – 242.0 452.2

5.3 8.7 – – – – – 55.9 251.0

Total concentration

Specification

52.4 124.1 108.5 130.1 161.2 99.4 – 531.0 1464.1

– – 100.0 120.0 150.0 100.0 – 550.0 1200.0–1400.0

Table 5 Measured concentrations for BAC homologues in samples using HPLC-UV (n = 3). Experimental concentration (␮g mL−1 )

Sample

Eye drops-1 Eye drops-2 Eye drops-3 Eye drops-4 Eye drops-5 Eye drops-6 Sanitizer-1 Sanitizer-2

Total concentration (␮g mL−1 )

C12 -BAC

C14 -BAC

C16 -BAC

36.6 76.5 103.6 127.8 148.9 70.6 228.4 736.7

11.1 36.4 – – – 21.1 235.4 433.1

– 10.1 – – – – 50. 4 241.9

47.9 123.0 103.6 127.8 148.9 91.7 514.2 1411.8

Table 6 Comparison of analysis performances of PS-MS and HPLC-UV. Method

Working ranges (␮g mL−1 )

Linear correlation coefficient

LOD (␮g mL−1 )

LOQ (␮g mL−1 )

RSD %

Analysis time (minutes)

PS-MS HPLC-UV

1–100 10–200

0.998–0.999 0.999–0.999

∼0.1 ∼3.0

<1 <10

0.7–5.5 0.3–3.6

2 25

homologues of benzalkonium chloride can be qualitatively and quantitatively analyzed. The qualitative analysis time is short (only 10 s), and can quickly screen antibacterial agents and obtain the composition of BAC homologues. Similarly, PS-MS can also give accurate quantitative analysis of BAC. High throughput, lower detection limit and quantitative limit characteristics that are desirable for quantitative methods are established in this paper, and

the method’s accuracy meets the exact quantitative claim. The detection limit and the quantitation limit are lower than those of HPLC-UV methods. The composition and content of BAC can be accurately determined. The analysis is simple, saves time and mobile phase costs, is more suitable for large quantities of market analysis and screening and is more environmentally friendly.

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