TW201447296A - A method of detecting paraben - Google Patents

Abstract

A method of detecting paraben, which is suitable for small amount of sample, is disclosed. The method of detecting paraben comprises: dissolving a sample by a solvent, followed by sonication and centrifugation to obtain a supernatant with paraben; adding a derivatizating agent to the supernatant and triggering a derivatization reaction between the supernatant and the derivatizating agent to obtain a derivatizating solution with tagged paraben; irradiating the tagged paraben by a laser bean to generate a gas ion, followed by detecting the mass-to-charge ratio of the gas ion, to obtain the molecular weight of the gas ion.

Description

Method for detecting parabens

The present invention relates to a detection method, and more particularly to a method for detecting the content of a paraben.

Referring to Fig. 1, para-hydroxybenzoic acid (paraben) includes methyl ester (methyparaben, MP), ethylparaben (EP), and propylparaben (PP). ), butylparaben (BP), isopropyl paraben (IPP), isobutylparaben (IBP) and secbutylparaben (SBP) are often added to cosmetics and pharmaceuticals. As a preservative, it can prevent mold and bacteria from growing, and can extend the life of the product. However, paraben preservatives are considered to have estrogen activity, affecting the male reproductive system, and are classified as xenoestrogen (XE) and endocrine-disrupting compounds (EDC). Also known as environmental hormones, it can cause harm to the human body; and, for individuals who are prone to cause allergic constitution, it is more likely to promote allergic reactions and photosensitivity. Therefore, parabens must be preservatives. The addition or not and its usage are strictly monitored.

The conventional method for detecting parabens is to first extract a sample to be tested by solid phase extraction (SPE) extraction membrane or extraction column, and then to reverse phase high pressure liquid layer. Regressed-phase high performance liquid chromatography (RP-HPLC) is combined with an ultraviolet detector (ultraviolet detector). Referred to as UV) or diode array detector (DAD) for analysis.

However, in the conventional method for detecting parabens, it is limited by the solid phase extraction method to separate and extract the sample to be tested, so a large amount of sample to be tested (0.2-20 g) is required, which is not applicable to It is more difficult to obtain, or a product with a higher price.

Moreover, in the conventional method for detecting parabens, reverse phase high pressure liquid chromatography not only analyzes the time longer, but after each sample analysis, additional time is required to rinse the column, and After the column is returned to equilibrium, the analysis of the next sample to be tested can be performed, which is quite time consuming and therefore not suitable for large-scale sample screening.

Furthermore, in the conventional method for detecting parabens, the reverse phase high pressure liquid chromatography system needs to rinse the chromatography column with a large amount of organic solvent, and therefore, a large amount of organic waste liquid is generated during the analysis. It can cause harm to the environment.

The main object of the present invention is to provide a method for detecting a paraben, which has a high degree of precision and can reduce the amount of the sample to be tested.

Still another object of the present invention is to provide a method for detecting a paraben which can greatly shorten the detection time to facilitate large-scale sample screening of a sample to be tested.

Another object of the present invention is to provide a method for detecting a paraben to reduce the discharge of organic waste liquid to avoid environmental pollution.

In order to achieve the foregoing object, the technical means and the effects that can be achieved by the technical method include: a method for detecting a paraben, comprising: a pre-treatment step, which is to be treated The sample is dissolved in a solvent, ultrasonically oscillated and centrifuged at high speed to obtain a supernatant containing a paraben; a chemical derivatization step is to add a derivatizing reagent to the supernatant a derivatization reaction of the derivatizing reagent with the paraben to obtain A derivatization solution is obtained, the derivatization solution comprising a p-hydroxybenzoate bonded to a mark; an extraction step of extracting the derivatization solution with an extraction solvent to obtain an extract containing the mark a para-hydroxybenzoate; and an analysis step of forming a gas phase ion by bonding a labeled para-hydroxybenzoate with a laser beam and detecting a mass-to-charge ratio of the gas phase ion by a mass analyzer To obtain the molecular weight of the gas phase ion.

The method for detecting a paraben of the present invention, wherein after the pretreatment step, before the chemical derivatization step, further comprises a temperature adjustment step of adjusting the temperature of the supernatant to 30-80 °C.

The method for detecting a parahydroxybenzoate of the present invention, wherein, in the chemical derivatization step, the derivatizing reagent has a chlorosulfonylphenyl group, and the chlorosulfonylphenyl group and the hydroxyl group of the parahydroxybenzoate are subjected to the Derivative reaction.

The method for detecting a parahydroxybenzoate of the present invention, wherein in the chemical derivatization step, the derivatizing reagent is 1,3-benzothiazole-6-sulfonyl chloride or 4-dimethylaminophenylazobenzene -4'-sulfonyl chloride or dansyl chloride.

The method for detecting a paraben of the present invention, wherein in the analyzing step, a laser beam having a wavelength of 330-360 nm is used to form the gas phase of the labeled para-hydroxybenzoate to form the gas phase. ion.

The method for detecting a parahydroxybenzoate of the present invention, wherein, in the pretreatment step, the sample to be tested is in the form of a liquid, an emulsion, a cream or a gel.

In the method for detecting a parahydroxybenzoate of the present invention, in the chemical derivatization step, an internal standard is further added to the supernatant, and the internal standard and the derivatizing reagent are subjected to the derivatization reaction.

The method for detecting a parahydroxybenzoate of the present invention, wherein, in the chemical derivatization step, the internal standard is an isotope of a paraben.

The method for detecting a parahydroxybenzoate of the present invention, via the chemical derivatization step The derivatization reaction of the chlorosulfonylphenyl group of the derivatizing reagent with the hydroxyl group of the parahydroxybenzoate enhances the identification efficiency and specificity of the para-hydroxybenzoate identification, and thus only a small amount of the sample to be tested is It can be accurately detected and is suitable for samples that are difficult to obtain or relatively expensive, and is an effect of the present invention.

The method for detecting a para-hydroxybenzoic acid ester of the present invention, wherein the labeled para-hydroxybenzoate is excited by a laser beam to form the gas phase ion, and according to the gas phase ion, The mass-to-charge ratio, and further whether the sample to be tested contains parabens, can greatly reduce the time of detection and analysis, and achieve the effect of rapid screening of the sample to be tested.

In the method for detecting the para-hydroxybenzoic acid ester of the present invention, only a small amount of the organic solvent can be used in the pre-treatment step and the extraction step, thereby reducing the amount of organic waste liquid generated during the detection process, thereby achieving the effect of reducing environmental pollution.

S1‧‧‧Pre-processing steps

S2‧‧‧Chemical Derivation Steps

S3‧‧‧ extraction step

S4‧‧‧ Analysis steps

S5‧‧‧ Temperature adjustment steps

Figure 1 is the structural formula of p-hydroxybenzoate.

Figure 2 is a flow chart of a first preferred embodiment of the present invention.

Figure 3 is a reaction formula of the derivatization reaction of the present invention.

Figure 4 is a flow chart of a second preferred embodiment of the present invention.

The above and other objects, features and advantages of the present invention will become more <RTIgt;

Referring to FIG. 1 , a method for detecting a parahydroxybenzoate according to the present invention comprises: a pretreatment step S1 of dissolving a sample to be tested in a solvent, ultrasonic vibration and high-speed centrifugation. Obtaining a supernatant, the supernatant containing a paraben; a chemical derivatization step S2, wherein a derivatizing reagent is added to the supernatant, and the derivatizing reagent is subjected to a derivatization reaction with the paraben to obtain a derivatized solution containing a p-hydroxy group bonded to a label. a benzoate; an extraction step S3, extracting the derivatization solution with an extraction solvent to obtain an extract containing a paraben bonded to the label; and an analysis step S4 A laser beam causes the labeled paraben to form a gas phase ion and analyzes the mass to charge ratio of the gas phase ion to obtain the molecular weight of the gas phase ion.

In detail, in the pre-treatment step S1, the sample to be tested may be selected from cosmetics or medicines that are relatively easy to contact in daily life, wherein the dosage form of the cosmetic may be selected as a solution or a lotion (lotion or Emulsion), cream or gel, and the drug may be an oral solvent, an oral lozenge, an injectable solvent, an inhalation solvent, a gel-like application agent or a cream-like application agent; The amount of the sample to be tested is selected to be between 0.1 and 100 mg. The sample to be tested is dissolved in the solvent, and the solvent may be selected from an organic solvent such as acetonitrile, acetone or methanol, or a solvent prepared by mixing the organic solvent and water in a specific ratio, in the preferred embodiment. The sample was selected to dissolve 100 mg of the sample to be dissolved in 1 ml of acetonitrile.

After the sample to be tested is dissolved in the solvent, the ultrasonic wave can be oscillated, so that the paraben in the sample to be tested can be completely dissolved in the solvent, and the ultrasonic oscillation can be performed for 1-30 minutes; The impurities of the sample to be tested are removed by high-speed centrifugation, and the high-speed centrifugation is preferably performed by centrifugation at a speed of 10,000-13,000 rpm for 1-30 minutes. In the preferred embodiment, the shock is oscillated for 10 minutes, so that the paraben in the sample to be tested can be completely dissolved in acetonitrile, and centrifuged at 13,000 rpm for 10 minutes at high speed to obtain the upper portion. The supernatant, and the supernatant contains a paraben.

In the chemical derivatization step S2, the derivatizing reagent is added to the supernatant, and the derivatizing reagent is subjected to the derivatization reaction with the paraben. Preferably, the derivatizing reagent has a wavelength of 330-360 nm. Absorbance value, and having a chlorosulfonylphenyl group (-SO ₂ Cl functional group), the chlorosulfonylphenyl group can be subjected to the derivatization reaction with a hydroxyl group of a parahydroxybenzoic acid ester (-OH functional group) to obtain the derivative A solution containing the p-hydroxybenzoate to which the label is attached (refer to the reaction formula shown in Fig. 3). The derivatization reagent may be 1,3-benzothiazole-6-sulfonyl chloride or 4-dimethylaminophenylazobenzene-4'-sulfonyl chloride (4). -dimethylaminoazobenzene-4'-sulfonyl chloride, also known as dabsyl chloride) or dansyl chloride, but not limited thereto.

Moreover, depending on the selected derivatizing reagent, the pH of the supernatant can be adjusted to facilitate the progress of the derivatization reaction. In the preferred embodiment, the derivatizing reagent is selected to be dansyl chloride, and the pH of the supernatant is adjusted to pH 8-10 with an alkaline solution (such as sodium carbonate or sodium hydrogencarbonate); Referring to FIG. 4, after the pre-treatment step S1, before the chemical derivation step S2, a temperature adjustment step S5 can be further performed, and the temperature of the supernatant can be adjusted to 30-80 ° C to accelerate. The derivatization reaction proceeds.

In the preferred embodiment, 5 μl of the supernatant is taken, the pH is adjusted to pH 8-10 with 10-500 mM sodium bicarbonate 10 μl, and 10 μl of dansyl chloride (dissolved in acetone, The concentration is 0.25-2 mg/ml) to obtain a mixed solution, and the mixed solution is heated to 30-80 ° C to accelerate the progress of the derivatization reaction, and the chlorosulfonylphenyl group (-SO ₂ Cl functional group) is The derivatization of the hydroxyl group of the paraben (-OH functional group) is carried out for 5 to 30 minutes, so that the p-hydroxybenzoate can be bonded to the label (as shown in Fig. 3) to obtain the derivatized solution.

In the extracting step S3, the derivatized solution may be extracted by using the extraction solvent to obtain the extract, the extract containing the p-hydroxybenzoate to which the label is attached; preferably, the extracting solvent may be The organic solvent is hexyl acetate, n-hexane, toluene or ethyl acetate. The extraction solvent is used in an amount of about 10-50 μl. In the preferred embodiment, the extraction is carried out with 20 μl of ethyl acetate. The labeled paraben can be dissolved in ethyl acetate to obtain the extract.

Matrix-assisted laser desorption ionization - analysis by a matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS), but not limited thereto. In detail, the extract may be placed on a target plate of the matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, covering a matrix to make the matrix and the extract Forming a crystalline solid; preferably, the matrix is selected to absorb α-cyanol-4-hydroxycinnamic acid (CHCA) having a wavelength of 330-360 nm. 3,5-dimethoxy-4-hydroxycinnamic acid (also known as sinapinic acid, abbreviated as SA) or dihydroxybenzoic acid (2,5-dihydroxybenzoic) Acid, abbreviated as 2,5-DHB), in the preferred embodiment, the matrix was selected to be 0.5 μl of α-cyano-4-hydroxycinnamic acid (concentration 10 mg/ml).

Then, the crystalline solid is irradiated with a laser beam having a wavelength of 330-360 nm, so that the crystalline solid can be excited to form the gas phase ions, and the gas phase ions can be accelerated into a mass after being accelerated by the electric field. An analyzer system for detecting a mass-to-charge ratio (m/z) of the gas phase ion to obtain a molecular weight of the sample to be tested; the mass analyzer may be selected from a flight time quality Time-of-flight analyzer (TOFanalyzer), quadrupole analyzer, ion trap analyzer (IT analyzer) and Fourier transform-ion cyclotron (Fourier transform-ion cyclotron) Resonance, referred to as FT-ICR). In the preferred embodiment, the mass analyzer is selected as a time-of-flight analyzer (TOF analyzer), which has a fast analysis speed, combined with a matrix-assisted laser desorption free method. Can effectively save time in sample analysis.

Preferably, in the chemical derivatization step S2, an internal standard is added to the supernatant, and the internal standard can be selected as an isotopes of parabens. In the examples, ethyl d5-p-hydroxybenzoate having a concentration of 16 μg/ml is selected, and the internal standard is also formed into the crystal with the matrix via the derivatization reaction. Solid matter, and excited by a laser beam to form the gas phase ions. The peak area of the internal standard is analyzed according to the mass analyzer, and the value is brought into the calibration curve of the paraben compared with the peak area obtained by the sample to be tested, that is, the sample in the sample to be tested can be calculated. The content of parabens.

In order to confirm that the preferred embodiment of the present invention can be used to detect the paraben content contained in the sample to be tested, several commercially available lotions, gelatinous cosmetics, emulsion cosmetics, creamy cosmetics, The mask and the clinical liquid medicine are detected and analyzed by the detection method of the paraben of the present invention, and the results are as shown in Table 1-6:

Please refer to the first table, the sample numbers 1-5 to be tested are respectively commercially available makeup. Water, 100 mg each, was tested for the parabens contained therein by the preferred embodiment of the invention (for methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and p-hydroxyl group, respectively). The content of butyl benzoate is determined to be 0.04-0.15% of para-hydroxybenzoate, respectively; accordingly, the detection method of the para-hydroxybenzoate of the preferred embodiment of the present invention is applicable to the detection electrode. A small amount (100 mg) of the sample to be tested.

Please refer to Table 2, sample numbers 6-7 to be tested are respectively commercially available gelatinous cosmetics, each taking 100 mg of the parabens contained therein according to the preferred embodiment of the present invention (respectively for the pair The content of methyl hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate was determined to contain 0.05-0.12% of para-hydroxybenzoate, respectively.

Referring to Table 3, the sample numbers 8-12 to be tested are respectively commercially available emulsion cosmetics, each of which is 100 mg. The p-hydroxybenzoate contained therein is detected by the preferred embodiment of the present invention (respectively for the pair The content of methyl hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate was found to contain 0.03-0.12% of para-hydroxybenzoate, respectively.

Referring to Table 4, sample numbers 13-17 to be tested are respectively commercially available cream-like cosmetics, each taking 100 mg of the parabens contained therein by the preferred embodiment of the present invention (respectively for The content of methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate was determined to contain 0.07-0.16% of para-hydroxybenzoate, respectively.

Please refer to Table 5, the sample numbers 18-22 to be tested are respectively commercially available mask products, and 100 mg of the essence added to the mask paper are respectively tested for the parabens contained therein by the preferred embodiment of the present invention. The content of the acid ester (for methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate) was found to contain 0.01-0.06% of p-hydroxybenzene respectively. Formate.

Please refer to Table 6, the sample numbers 23-27 to be tested are respectively commercially available clinical liquid medicines, each taking 100 mg to detect the p-hydroxy group contained therein by the preferred embodiment of the present invention. The content of benzoic acid ester (detected for methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate) was found to contain 0.01-0.23% of the pair respectively. Hydroxybenzoate.

In addition, another commercially available lotion, gelatinous cosmetic, emulsion cosmetic, creamy cosmetic, facial mask and clinical liquid pharmaceutical, each taking 0.1 mg, is tested for the p-hydroxybenzene contained therein by the preferred embodiment of the present invention. The content of formate (detected for methylparaben, ethylparaben, propylparaben and butylparaben) was as shown in Table 7.

MP: methyl p-hydroxybenzoate, EP: ethyl p-hydroxybenzoate, PP: propyl p-hydroxybenzoate, BP: butyl p-hydroxybenzoate

Please refer to the seventh table, the sample numbers 28-33 to be tested are commercially available lotion, gelatinous cosmetic, emulsion cosmetic, creamy cosmetic, facial mask and clinical liquid pharmaceutical, each taking 0.1 mg via the present invention. The preferred embodiment detects the content of the parabens contained therein (for methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and butyl p-hydroxybenzoate, respectively), It is found that 0.13-2.20% of the parabens are respectively contained, and the analysis results are consistent with the test results of 100 mg of the sample to be tested, indicating the detection of the paraben of the preferred embodiment of the present invention. The method is also suitable for the detection of very small amounts (0.1 mg) of samples.

According to the test results of Tables 1-7, the method for detecting the parahydroxybenzoate of the present invention is directed to methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate and dibutylparaben. The esters all have a good linear relationship with a quantitative range of 0.1-10 μg/ml, a coefficient of determination (R ² ) ≧0.9995, and a relative standard deviation of <9.86%. Further, the method for detecting a parahydroxybenzoate of the present invention can be further applied to detect a sample to be tested of a different texture such as a liquid, a gel, an emulsion or a cream.

In summary, the method for detecting a parahydroxybenzoate of the present invention, through the chemical derivatization step, the derivatization reaction of the chlorosulfonylphenyl group of the derivatizing reagent with the hydroxyl group of the paraben to enhance the paraben The identification efficiency and specificity of the acid ester are recognized, so that only a small amount of the sample to be tested (0.1-100 mg) can be accurately detected, and is suitable for the sample to be tested which is difficult to obtain or relatively expensive, which is the present invention. efficacy.

Furthermore, the method for detecting a paraben of the present invention, through the analyzing step, can form the gas phase ion by bonding the labeled paraben, and detecting the gas according to a mass analyzer. The mass-to-charge ratio of the phase ions, and further whether the sample to be tested contains parabens, can greatly reduce the time of detection and analysis, and achieve the effect of rapid screening of the sample to be tested.

Moreover, the method for detecting the parahydroxybenzoate of the present invention can reduce the amount of organic waste liquid generated during the detection process by using a small amount of the organic solvent only in the pretreatment step and the extraction step, thereby achieving the effect of reducing environmental pollution. .

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

S1‧‧‧Pre-processing steps

S2‧‧‧Chemical Derivation Steps

S3‧‧‧ extraction step

S4‧‧‧ Analysis steps

Claims (8)

A method for detecting a paraben, comprising: a pretreatment step of dissolving a sample to be tested in a solvent, ultrasonically oscillating and high-speed centrifugation to obtain a supernatant, the supernatant system Containing a paraben; a chemical derivatization step, adding a derivatizing reagent to the supernatant, and derivatizing the derivatizing reagent with the paraben to obtain a derivatized solution, the derivatizing solution a step of extracting a labeled paraben; an extraction step of extracting the derivatized solution with an extraction solvent to obtain an extract comprising a paraben bonded to the label; and The analyzing step is a laser beam that causes the labeled paraben to form a gas phase ion, and the mass-to-charge ratio of the gas phase ion is detected by a mass analyzer to obtain the molecular weight of the gas phase ion. The method for detecting a paraben according to claim 1, wherein after the pretreatment step, before the chemical derivatization step, a temperature adjustment step is further included to adjust the temperature of the supernatant. Adjust to 30-80 °C. The method for detecting a paraben according to claim 1 or 2, wherein in the chemical derivatization step, the derivatizing reagent has a chlorosulfonylphenyl group, and the chlorosulfonylphenyl group and the pair The hydroxyl group of the hydroxybenzoate undergoes the derivatization reaction. The method for detecting a paraben according to claim 3, wherein in the chemical derivatization step, the derivatizing reagent is 1,3-benzothiazole-6-sulfonyl chloride or 4-dimethyl Aminophenylazobenzene-4'-sulfonyl chloride or dansyl chloride. The method for detecting a paraben according to claim 1 or 2, wherein in the analyzing step, the laser is bonded to the labeled hydroxyl group by a laser beam having a wavelength of 330-360 nm. The benzoate forms the gas phase ion. The method for detecting a paraben as described in claim 1 or 2, Wherein, in the pre-treatment step, the sample to be tested is in the form of liquid, emulsion, cream or gel. The method for detecting a paraben according to claim 1 or 2, wherein in the chemical derivatization step, an internal standard is added to the supernatant to make the internal standard and the internal standard The derivatization reagent performs the derivatization reaction. The method for detecting a paraben according to item 8 of the patent application, wherein in the chemical derivatization step, the internal standard is an isotopes of a paraben.