A method for detecting phthalates in milk based on Au@Ag@IP6 SERS substrate

Abstract

This invention belongs to the field of food safety testing technology, specifically disclosing a method for detecting phthalic acid esters (PAEs) in milk based on an Au@Ag@IP6 SERS substrate. The method includes the following steps: preparing gold nanoparticles; preparing Au@Ag@IP6 nanoparticles; preparing an Au@Ag@IP6NPs SERS substrate; preparing phthalohydrazide standards and phthalic acid ester standards; preparing phthalohydrazide generated from the reaction of phthalic acid esters; preparing milk samples with the standard and pretreatment; preparing the SERS sample; and Raman detection. This invention can uniformly convert more than thirty types of PAEs into phthalohydrazide molecules, solving the problem of PAEs' water insolubility and enabling simultaneous detection of the total amount of all types of PAEs. Compared with detection methods using other gold or silver nanoparticles as SERS substrates, this method can specifically capture phthalohydrazide molecules, resulting in higher sensitivity and a lower detection limit. After preparing the SERS substrate, only simple pretreatment is required to detect PAEs, enabling rapid detection of PAEs in milk within 15 minutes.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate. Background Technology

[0002] Phthalate esters (PAEs) are a class of plasticizers that are widely used in the medical, industrial and human life fields [1]. They are often added to products such as food packaging, toys, cosmetics and medical devices to make plastics more elastic and improve their strength and durability. The global annual consumption of PAEs is close to 100,000 tons. China is the largest plasticizer market, producing more than 45,000 tons of PAEs annually, accounting for 45% of the global total. However, PAEs are extremely harmful to the human body. They enter the human body through breathing, oral cavity, skin contact, etc., are adsorbed by solid particles, and eventually accumulate in the body. PAEs have been detected in human tissues and secretions (such as lungs, colon, breast milk and placenta). PAEs are environmental estrogens with reproductive and developmental toxicity. They have potential carcinogenic, teratogenic, and mutagenic toxicity, which can cause systemic dysfunction, affect the endocrine system, affect the development of the nervous system in newborns, cause precocious puberty in girls, and may affect embryonic development and uterine mucosal tissue hyperplasia through placental lipid and zinc metabolism. At the same time, they have a great impact on the developing male reproductive system [5]. There are many types of PAEs, more than dozens. In China, only four types of PAEs, namely di(2-ethylhexyl) phthalate (DEHP), diallyl phthalate (DAP), diisononyl phthalate (DINP), and dibutyl phthalate (DBP), are allowed to be used in food packaging. PAEs should be prohibited in oily foods and infant foods [6]. The maximum residue limits of DEHP, DINP, and DBP are 1.5, 9.0, and 0.3 mg·kg-1, respectively. However, due to the fat-soluble nature of PAEs, they can still migrate from plastics into food, especially into packaged milk. DBP and DEHP are the two most prominent types in packaged milk, accounting for approximately 47% of the total PAE content in milk. Therefore, it is crucial to develop a simple and rapid method for detecting PAE molecules in packaged milk.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique that provides rich chemical fingerprint information of analytes at the single-molecule scale. Since Fleischmann et al. discovered in 1974 that the Raman signal of pyridine molecules adsorbed on a rough silver electrode surface was significantly enhanced, SERS technology has developed rapidly. When molecules adsorb onto rough metal surfaces (such as silver, copper, and gold), the plasmon energy on the metal surface is excited to a high energy level due to illumination. This plasmon energy couples with the electric field of the light wave and resonates, resulting in a significant enhancement of the local electromagnetic field on the metal surface. This molecular signal on a rough silver surface is up to 106 times stronger than in solution, making the originally weak Raman signal detectable. SERS technology not only has simple pretreatment but also offers advantages such as high sensitivity, high selectivity, non-destructive nature, and rapid analysis, making it widely applicable in many fields, including biomedicine, environmental monitoring, and food safety. Current methods for detecting phthalate (PAEs) include traditional analytical methods such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and high-performance liquid chromatography (HPLC). These methods offer high sensitivity and accuracy, but their pretreatment processes are quite complex. Furthermore, using biosensor technology for selective identification and quantitative analysis of PAEs requires regular sensor replacement and maintenance, which is time-consuming and labor-intensive.

[0004] Therefore, it is essential to develop a low-cost, simple, rapid, and highly sensitive method for detecting PAEs in packaged milk. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for detecting phthalate (PAEs) in packaged milk based on an Au@Ag@IP6 SERS substrate. By using Au@Ag@IP6 as the SERS substrate, the strong metal complexation between IP6 and metal nanoparticles, as well as the hydrogen bonding between IP6 and phthalohydrazide (PHAH2), tightly adsorbs phthalohydrazide molecules into the "hot spot" region of the metal nanoparticles, enabling quantitative detection of PHAH2 and thus indirectly detecting PAEs. This research provides a new diagnostic approach for determining whether plasticizers exceed standards in milk. More importantly, it holds promise as a universal technology for the accurate detection of PAEs in other systems such as edible oils and urine.

[0006] To achieve the above objectives, one of the technical solutions of the present invention is: a method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate, comprising the following steps:

[0007] S1: Preparation of gold nanoparticles;

[0008] S2: Preparation of Au@Ag@IP6 nanoparticles: Using the gold nanoparticles prepared in step S1 as seeds, Au@Ag nanoparticles (Au@Ag@IP6 NPs) capped with inositol hexaphosphate (IP6) were prepared.

[0009] S3: Preparation of Au@Ag@IP6 NPs SERS substrate: The Au@Ag@IP6 NPs prepared in step S2 are evenly spread on the gold sheet and dried;

[0010] S4: Preparation of phthalohydrazide standards and phthalic acid ester (PAE) standards;

[0011] S5: Preparation of phthalic acid hydrazide generated by the reaction of phthalic acid esters (PAEs): The phthalic acid ester (PAEs) standard obtained in step S4 is diluted and then hydrazine hydrate is added. The reaction is carried out under water bath conditions to obtain phthalic acid hydrazide.

[0012] S6: Preparation and pretreatment of milk standard: Add the phthalate (PAEs) standard obtained in step S4 to the milk to prepare the milk standard. Add ethanol to the milk standard, centrifuge by sonication, take the supernatant, blow dry through a membrane, add deionized water to reconstitute, extract the phthalates with n-hexane, add ethanol to reconstitute the phthalates, and then add hydrazine hydrate to react under water bath conditions.

[0013] S7: Preparation of SERS test samples: Different concentrations of phthalohydrazide standards, different concentrations of phthalohydrazide generated by the reaction of phthalic acid esters (PAEs), and phthalic acid esters (PAEs) in milk system were spiked onto the Au@Ag@IP6 NPs SERS substrate prepared in S3 to simulate detection.

[0014] S8: Raman detection: Raman detection is performed on the SERS sample obtained in step S7, and the measured SERS spectra are compared and analyzed.

[0015] The analysis revealed characteristic peaks at 712, 1026, and 1376 cm⁻¹, which can be attributed to CN vibration, aromatic ring vibration, and ring stretching vibration of 2,3-diazanaphthalene compounds, respectively. The results indicate that by using hydrazine hydrate to convert phthalic acid esters (PAEs) in milk into phthalohydrazides, quantitative detection of phthalic acid esters (PAEs) in milk can be achieved.

[0016] In a preferred embodiment of the present invention, the gold nanoparticle preparation method in step S1 is as follows: 150-250 mL of chloroauric acid aqueous solution (0.05-0.015 wt%) is boiled for 5-15 min, then 1-3 mL of sodium citrate (0.5-0.15 wt%) is added, and the mixture is stirred continuously for 10-20 min. After cooling to room temperature, gold nanoparticles with a particle size of 35-55 nm and an ultraviolet absorption wavelength mainly in the range of 500-550 nm are obtained.

[0017] In a preferred embodiment of the present invention, the preparation method of Au@Ag@IP6 NPs in step S2 includes taking 4-7 mL of 15-25 mM silver nitrate aqueous solution and adding 7-10 mL of 0.5-1.5 mM IP6 aqueous solution; taking 50-60 mL of deionized water and adding 5-15 mL of gold nanoparticles prepared in step S1, while stirring, adding 2-3 mL of 0.5-1.5 wt% sodium citrate and 0.5-1.5 wt% ascorbic acid, and adding 8-12 mL of AgNO3 at a stirring rate of 160-220 μL / min to obtain Au@Ag@IP6 NPs with a particle size of 100-200 nm.

[0018] The role of IP6 is to complex with strong metals to form an ultrathin coating on the Ag shell, which is used to adsorb phthalohydrazide molecules and obtain super strong SERS enhancement.

[0019] In a preferred embodiment of the present invention, the gold sheet in step S3 needs to be soaked in acetone for 20-40 minutes, then soaked in piranha solution for 20-40 minutes to remove organic matter, and finally rinsed several times with ultrapure water until clean.

[0020] In a preferred embodiment of the present invention, the preparation method of the phthalic acid hydrazine standard in step S4 is to dilute the phthalic acid hydrazine with ethanol and water at a volume ratio of 1:1 to a concentration of 0.1-1000 mg / L. -1 Phthalohydrazide standards, the preparation method of phthalic acid ester (PAE) standards is to dilute the stock solution of phthalic acid ester (PAE) standards with ethanol to a concentration of 0.1-1000 mg / L. -1 .

[0021] In a preferred embodiment of the present invention, in step S5, the volume ratio of phthalic acid ester (PAE) standard to hydrazine hydrate is (3-5):1, the water bath temperature is 75-85°C, and the reaction time is 5-15 min.

[0022] PAEs are reacted to phthalic acid hydrazide in ethanol. The insoluble phthalic acid esters are converted into water-soluble phthalic acid hydrazide using hydrazine hydrate. The converted phthalic acid hydrazide is then adsorbed onto the surface of Au@Ag@IP6NPs and enters the "SERS hotspot".

[0023] In a preferred embodiment of the present invention, step S6 simulates the environment in which plastic migrates into the milk by spiked milk.

[0024] In a preferred embodiment of the present invention, the signal molecule detected by Raman in step S7 is phthalic acid hydrazide.

[0025] In a preferred embodiment of the present invention, step S7 uses a portable Raman spectrometer or other type of Raman spectrometer.

[0026] In a preferred embodiment of the present invention, the excitation wavelength of Raman detection in step S7 is 785 nm, the power is 500 mW, and the time is 150 ms.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention constructs a platform for detecting phthalates in milk based on SERS, wherein the Au@Ag@IP6NPs SERS substrate provides SERS "hot spots" for more compact adsorption of phthalohydrazide molecules, making this invention fast, convenient, highly sensitive and highly specific, and capable of fingerprint recognition. This method can achieve rapid detection of phthalate molecules in milk within 15 minutes.

[0029] 2. This invention can uniformly convert more than 30 types of phthalates into phthalohydrazide, which not only solves the problem of the non-water solubility of phthalate esters, but also enables the simultaneous detection of multiple types of phthalates and the detection of the total amount of all types of phthalates.

[0030] 3. Compared with the detection method using gold sol as SERS substrate, the present invention has higher detection sensitivity and lower detection limit, and can better capture phthalic acid hydrazide molecules. After preparing the SERS substrate, only simple pretreatment is required to detect phthalic acid ester, realizing rapid measurement of phthalic acid ester Raman signal in a short time. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the principle of the present invention;

[0032] Figure 2 This is a comparison of the surface-enhanced Raman spectra of phthalohydrazide standard and phthalohydrazide derived from PAEs standard;

[0033] Figure 3 The concentration is 10 μg / L -1 50 μg L -1 100 μg L -1 150 μg L -1 200 μg L -1 Surface-enhanced Raman spectra of phthalohydrazide standards;

[0034] Figure 4 For a concentration of 10 μg / L -1 50 μg L -1 100 μg L -1 150 μg L -1 200 μg L -1 Surface-enhanced Raman spectra of phthaloyl hydrazine generated by reacting a mixture of six phthalate standards with hydrazine hydrate;

[0035] Figure 5 The concentrations of the mixed sample of six phthalates before and after the reaction were measured at 1376 cm⁻¹. -1 A graph showing the relationship between peak area and peak area;

[0036] Figure 6 The actual milk system was spiked with a concentration of 100 μg / L. -1 Six different PAEs and 100 μg L -1 Surface-enhanced Raman spectra of a mixture of six PAEs and a blank milk sample;

[0037] Figure 7 These are surface-enhanced Raman spectra of a mixture of six PAEs at different concentrations in an actual milk system. Detailed Implementation

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0039] Example 1

[0040] Preparation of gold nanoparticles (AuNPs): 200 mL of chloroauric acid aqueous solution (0.01 wt%) was added to a 250 mL round-bottom flask and stirred until boiling. After boiling for 10 min, 2 mL of sodium citrate (1 wt%) was added and stirred for 15 min.

[0041] Preparation of Au@Ag@IP6 nanoparticles (Au@Ag@IP6NPs): IP6-capped Au@Ag nanoparticles were prepared. Silver nitrate aqueous solution was prepared in advance. 5.6 mL of 20 mM silver nitrate aqueous solution was transferred to a 15 mL centrifuge tube, and then 8.4 mL of 1 mM IP6 aqueous solution was added. First, 55 mL of Milli-Q water was taken, and 10 mL of gold seeds prepared with S1 at 45 nm were added. While stirring, 2.4 mL of sodium citrate (1 wt%) and ascorbic acid (1 wt%) were added. The prepared silver nitrate solution was then added to a round-bottom flask using a stepper motor at a rate of 200 μL / min. The mixture was stirred until all nitrates were added, resulting in Au@Ag@IP6NPs with a particle size of 150 nm.

[0042] Preparation of Au@Ag@IP6NPs SERS substrate: Take 5 mL of the prepared 150 nm Au@Ag@IP6NPs, centrifuge at 3500 rpm for 5 minutes, remove the supernatant, wash once with 1 mM IP6, and finally adjust the volume to 100 μL. Take 3 μL onto a gold sheet and let it air dry to obtain a uniform SERS substrate;

[0043] Preparation of phthalohydrazide standard and six different PAEs standards: The glass bottle was washed with ethanol and dried. 20 mg of phthalohydrazide powder was weighed into a glass bottle, and 20 mL of ethanol and water (ethanol:water = 1:1) was added to prepare a 1000 ppm standard. This standard was then serially diluted to 100 mg / L. -1 10mg L -1 1mg L -1 100 μg L -1 10 μg L -1 Six PAEs standards were prepared: di(2-ethylhexyl) phthalate (DEHP), diallyl phthalate (DAP), diisononyl phthalate (DINP), dibutyl phthalate (DBP), di-n-octyl phthalate (DNOP), dicyclohexyl phthalate (DCHP), and dihexyl phthalate (DHP). 20 mg of each stock solution was weighed into six glass vials, and 20 ml of ethanol was added to each vial to prepare 1000 mg L / L solutions. -1 The standard was then serially diluted to 100 mg / L. -1 10mg L -1 1mg L -1 100 μg L -1 10 μg L -1 Weigh out 3 mg of each of the six PAEs standards, mix them, and place them in a glass bottle. Add 18 ml of ethanol to prepare a 1000 mg / L solution.-1 The standard mixture was then serially diluted to 100 mg / L. -1 10mg L -1 1 mg / L -1 100 μg L -1 10 μg L -1 .

[0044] React PAEs to phthalic acid hydrazide: Take 10 μg L of the above concentrations respectively. -1 50 μg L -1 100 μg L -1 150 μg / L -1 200 μg L -1 One mL of each of the six PAE standard solutions was placed into five 3 mL glass bottles, and 250 μL of hydrazine hydrate was added to each bottle. The mixtures were reacted in a water bath at 60 °C for 10 minutes to obtain phthalic acid hydrazide.

[0045] Preparation and pretreatment of milk spiked sample: Prepare 50 μg L -1 100 μg L -1 150 μg L -1 Add standard to milk (weigh 0.2g of milk, add 10μL, 20μL, and 30μL of 1mg L-10 ... -1 (Prepared from a mixture of PAEs standards) to simulate the environment of plasticizer migration into milk. The milk was pretreated by adding the standard: 4 mL of ethanol was added, sonicated for 10 min, centrifuged at 5000 rpm / min for 2 min, the supernatant was collected, filtered through a membrane, dried, reconstituted with 1 mL of deionized water, extracted with 400 μL of n-hexane, dried, reconstituted with 1 mL of ethanol, and then 250 μL of hydrazine hydrate was added. The mixture was then incubated in a 60℃ water bath for 10 minutes.

[0046] Preparation of SERS test samples: 3 μL of phthalic acid hydrazide standards of different concentrations and phthalic acid hydrazide of different concentrations derived from PAEs reaction and phthalic acid esters in the milk system of the above preparation were added to the Au@Ag@IP6NPs SERS substrate to simulate detection.

[0047] The prepared SERS sample was subjected to Raman detection using a portable Raman detector with an excitation wavelength of 785 nm, a power of 500 mW, and a duration of 150 ms. The measured SERS spectra were then compared and analyzed.

[0048] 100μg L -1 SERS spectra of phthalic acid hydrazine derived from PAEs reaction and phthalic acid hydrazine standard at this concentration ( Figure 2 Comparative analysis revealed that they were all at 712cm.-1 1026cm -1 and 1376cm -1 Characteristic spectral peaks appear at these locations, which can be attributed to CN vibration, aromatic ring vibration, and ring stretching vibration peaks of 2,3-diazanaphthalene compounds, respectively.

[0049] The results showed that by using hydrazine hydrate to convert PAEs in milk into phthalic acid hydrazine, quantitative detection of PAEs in milk can be achieved.

[0050] The schematic diagram of surface Raman spectroscopy testing of phthalohydrazide molecules is shown below. Figure 1 As shown; for 100 μg L -1 Surface-enhanced Raman spectroscopy was performed on phthalohydrazide, a product of the reaction of a mixture of six different phthalic acid ester standards with hydrazine hydrate, and phthalohydrazide standards of the same concentration. The results are as follows: Figure 2 As shown; for a concentration of 10 μg / L -1 50 μg L -1 100 μg / L -1 150 μg L -1 200 μg L -1 Surface-enhanced Raman spectroscopy was performed on the phthalohydrazide standard, and the results are as follows: Figure 3 As shown; for a concentration of 10 μg / L -1 50 μg L -1 100 μg L -1 150 μg L -1 200 μg L -1 The phthalic acid hydrazide produced by reacting a mixture of six phthalic acid ester standards with hydrazine hydrate was subjected to surface-enhanced Raman spectroscopy, and the results are as follows: Figure 4 As shown; the concentrations of the mixed sample of six phthalates before and after the reaction at 1376 cm⁻¹ -1 The relationship between peak area and peak area is shown in the figure below. Figure 5 As shown; 100 μg / L was spiked into the actual milk system. -1 Surface-enhanced Raman spectroscopy was performed on six different phthalates, their mixtures, and a milk blank sample. The results are as follows: Figure 6 As shown; for milk spiked at a concentration of 50 μg / L -1 100 μg L -1 and 150 μg L -1 Surface-enhanced Raman spectroscopy was performed on a mixture of six phthalate esters, and the results are as follows: Figure 7 As shown.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate, characterized in that, Includes the following steps: S1: Preparation of gold nanoparticles; The method for preparing gold nanoparticles is to boil 150-250 mL of chloroauric acid aqueous solution (0.05-0.015 wt%) for 5-15 min, then add 1-3 mL of sodium citrate (0.5-0.15 wt%), stir continuously for 10-20 min, and cool to room temperature to obtain gold nanoparticles with a particle size of 50-60 nm and an ultraviolet absorption wavelength of 500 nm-550 nm. S2: Preparation of Au@Ag@IP6 nanoparticles: Using the gold nanoparticles prepared in step S1 as seeds, Au@Ag nanoparticles (Au@Ag@IP6 NPs) capped with inositol hexaphosphate are prepared. The preparation method of Au@Ag@IP6 NPs includes taking 4-7 mL of 15-25 mM silver nitrate aqueous solution and adding 7-10 mL of 0.5-1.5 mM IP6 aqueous solution; taking 50-60 mL of deionized water and adding 5-15 mL of the gold nanoparticles prepared in step S1, while stirring, adding 2-3 mL of 0.5-1.5 wt% sodium citrate and 0.5-1.5 wt% ascorbic acid, and adding 8-12 mL of AgNO3 at a stirring rate of 160-220 μL / min to obtain Au@Ag@IP6 NPs with a particle size of 100-200 nm. S3: Preparation of Au@Ag@IP6 NPs SERS substrate: The Au@Ag@IP6 NPs prepared in step S2 are evenly spread on the gold sheet and dried; S4: Preparation of phthalohydrazide and phthalic acid ester standards; S5: Preparation of phthalic acid hydrazide generated by the reaction of phthalic acid esters: The phthalic acid ester standard obtained in step S4 is diluted and then hydrazine hydrate is added. The reaction is carried out under water bath conditions to obtain phthalic acid hydrazide. S6: Preparation and pretreatment of milk standard: Add the phthalate standard obtained in step S4 to the milk to prepare the milk standard. Add ethanol to the milk standard, centrifuge by sonication, take the supernatant, blow dry through a membrane, add deionized water to reconstitute, extract the phthalate with n-hexane, add ethanol to reconstitute the phthalate, and then add hydrazine hydrate to react under water bath conditions. S7: Preparation of SERS test samples: Phthalohydrazide standards of different concentrations, phthalohydrazide generated from the reaction of phthalic acid esters of different concentrations, and phthalic acid esters in milk system were spiked onto the Au@Ag@IP6 NPs SERS substrate prepared in S3 to simulate detection. S8: Raman detection: Raman detection is performed on the SERS sample obtained in step S7, and the measured SERS spectra are compared and analyzed.

2. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, In step S3, the gold sheet needs to be soaked in acetone for 20-40 minutes, then in piranha solution for 20-40 minutes to remove organic matter, and finally rinsed several times with ultrapure water until clean.

3. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, The preparation method of the phthalohydrazide standard in step S4 is to dilute the phthalohydrazide with ethanol and water at a volume ratio of 1:1 to a concentration of 0.1-1000 mg / L. -1 Phthalohydrazide standards are prepared by diluting the stock solution of phthalic acid ester standards with ethanol to a concentration of 0.1-1000 mg / L. -1 .

4. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, In step S5, the volume ratio of phthalate standard to hydrazine hydrate is (3-5):

1.

5. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, The water bath temperature in steps S5 and S6 is 75-85℃, and the reaction time is 5-15 min.

6. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, The signal molecule detected by Raman in step S7 is phthalic acid hydrazide.

7. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, In step S7, Raman detection is performed using a portable Raman spectrometer or other types of Raman spectrometers.

8. The method for detecting phthalates in milk based on an Au@Ag@IP6 SERS substrate as described in claim 1, characterized in that, In step S7, the excitation wavelength for Raman detection is 785 nm, the power is 500 mW, and the time is 150 ms.

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