The proteases in cow’s milk have a great influence on the flavor and quality, especially on the shelf life of dairy products, but there is no uniform reference standard for the detection of protease activity in dairy products. This paper elaborates the sources, types and characteristics of protease in cow’s milk, and summarizes recent progress in the detection of the activity of different types of protease in cow’s milk, pointing out that the precise detection of protease activity in milk has great significance for dairy production and quality assurance.

Abnormal analysis of trace elements and heavy metal over-standard in dairy products are major concerns in the dairy industry, and how to detect elements of concern in dairy products quickly and accurately has become the focus of attention. Detection of elements in milk and dairy products is a prerequisite to ensure product quality and safety. Therefore, the pretreatment and detection methods specified in the Chinese and international standards for elements in milk and dairy products are reviewed. The principle, pros and cons of each method are elaborated so as to provide a reference for the detection of elements in the dairy industry.

A method for the determination of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside F, dulcoside A, rubusoside and steviolbioside in fermented milk by high performance liquid chromatography (HPLC) was developed and validated. The chromatographic conditions and sample pretreatment conditions were optimized. Samples were ultrasonically extracted with water for 20 min and for another 10 min after addition of 30% acetonitrile in water bath at 45 ℃. Potassium ferrocyanide and zinc acetate were added to precipitate proteins. The chromatographic separation was achieved on a C18 with gradient elution using a mobile phase consisting of sodium phosphate buffer and acetonitrile, and the analytes were detected by an ultraviolet (UV) detector at 210 nm. The results showed that the method was linear in the concentration range of 1–50 μg/mL, the limit of detection (LOD, RS/N = 3) was 3.0 mg/kg, and the limit of quantitation (LOQ, RS/N = 10) was 10.0 mg/kg. The average recoveries of all analytes spiked at 15–200 mg/kg were between 92.0% and 103.1%, with relative standard deviation (RSDs) (n = 6) less than 3.0%. This method was rapid, accurate, stable, sensitive, and suitable for the analysis of the content of steviol glycosides in fermented milk.

Sample pretreatment is a key step in element testing, which directly affects the precision and accuracy of analytical results. In order to meet the requirements for the detection of various elements, in this study, we used microwave digestion combined with inductively coupled plasma-mass spectrometry (ICP-MS) operated in the collision reaction mode to determine the content of multiple elements in milk powder, and investigated the influence of sample pretreatment conditions (acid removal time, sample size, and the amount of residual acid) on the detection results. Compared to removing acid mist only, better results were obtained under the following conditions: acid removal temperature was 160 ℃, acid removal time was 97.5 min, and sample size was 0.250 0 g, and the amount of residual acid was 1.0 mL. This pretreatment process is characterized by good stability, high precision and high accuracy.

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to measure the aluminum content in the quality control sample of milk powder, and an X-control chart was established for runaway judgement and handling during the operation process. The precision and median line of the control chart for new data points obtained one year later were evaluated using Minitab software with test for equal variance and analysis of variance. The standard deviation for the previous 25 data points was 0.186 mg/kg compared to 0.126 mg/kg for the 35 new ones, while the median values of the two groups did not differ from each other. More reliable control limit was calculated based on all the data points, and the new median value was 1.334 mg/kg with a standard deviation of 0.154 mg/kg. The decrease in standard deviation suggested that the precision was improved with smaller data fluctuations. In conclusion, calculation of new and more reliable control limit from more data is useful to find obvious systematic errors, abnormal data and slow changing trend in a timely and effective way and can provide evidence to decide whether data should be retained or removed so as to directly reflect the stability and tendency of the detection.

A spectrophotometric method for the determination of tea polyphenols in milk tea powder was developed by using Folin-phenol reagent. Crucial operating parameters such as extraction reagent, number of extraction cycles, temperature and derivatization reagent were investigated. Extraction using 70% methanol solution as the extraction reagent in a hot water bath at 70 ℃ performed twice and oxidization of the resulting extract with Folin-phenol reagent proved optimal. The detection wavelength was selected as 765 nm, and gallic acid was used as the calibration standard for the quantitation of tea polyphenols. The recoveries for whole milk powder spiked at concentration levels of 850–4 500 mg/kg were in the range of 98.9%–104.9%. Good stability and accuracy were achieved. When milk tea powder samples with different contents of tea polyphenols were detected in six replicates, relative standard deviations (RSDs) of 0.29%–0.84% were obtained, indicating good precision. This method proved to be accurate, easy to operate and repeatable with simple and easily available instruments, and thus suitable for the analysis of tea polyphenols in milk tea powder.

A reversed-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantitative determination of vanillin, methyl vanillin, ethyl vanillin and coumarin in milk and dairy products has been established. Vanillin compounds from samples were extracted with acetonitrile, blown to dryness under nitrogen, and then cleaned up with n-hexane. The chromatographic separation was achieved using a mixture of 20 mmoL/L ammonium acetate (pH 5.6), acetonitrile and methanol as the mobile phase with gradient elution on a C18 column. The analysis was carried out using a UV detector at a wavelength of 267 nm and the analytes were quantified by an external standard method. Good linear relationships in the range of 0.05–5.00 μg/mL were observed for all analytes. The limit of quantification was 0.2 mg/kg and the limit of detection was 0.06 mg/kg for the four analytes in liquid milk, milk powder, yogurt, lactic acid fermented beverage, ice cream, and cheese. The recoveries for vanillin, methyl vanillin, ethyl vanillin and coumarin in these samples at spiked concentration levels of 0.2–2.0 mg/kg were 80.6%–110.0%, 80.1%–109.6%, 80.4%–109.5% and 80.1%–105.9%, with relative standard deviations of 1.87%–7.70%, 1.45%–9.80%, 1.66%–9.52% and 1.16%–9.52%, respectively. This method was rapid, accurate, repeatable and simple and could be applied to determine vanillin compounds in milk and dairy products.

A method for the determination of lactose in low-lactose milk and lactose-free milk by using ion chromatography was developed. Lactose from samples was extracted with 3% acetic acid as a protein precipitator. The method was performed on a CarboPacTM PA20 column by gradient elution. The analyte was detected with an electrochemical detector and quantified by an external standard method. The limit of quantification was 100 mg/kg and the limit of detection was 50 mg/kg for lactose in milk. The recoveries at spiked concentration levels of 100–500 mg/kg were 92.3%–103.4%, with relative standard deviations between 1.44% and 4.43%. This method proved to be rapid, accurate, repeatable, sensitive and simple.

A method for the determination of deoxynivalenol (DON) and its derivatives 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) in dairy products, cereal flour and dairy cow feed was developed using high performance liquid chromatography (HPLC). Samples were extracted with ultrapure water, and the extract was cleaned up by immunoaffinity column chromatography and detected by HPLC with ultraviolet detection (HPLC-UVD). The limit of quantitation of the method was 100 μg/kg for all three analytes in dairy products, cereal flour and feed. Recoveries from these three sample matrices spiked at concentration levels ranging from 100 to 500 μg/kg were higher than 90% for DON and higher than 80% for 3-ADON and 15-ADON. This method proved to be simple, fast, sensitive, highly selective, and suitable for the simultaneous analysis of DON, 3-ADON and 15-ADON in dairy products, cereal flour and feed.

An high performance liquid chromatographic (HPLC) method for the determination of four sweeteners and one preservative in fermented milk and jam was developed in this paper. Water was used as the extraction solvent. The chromatographic separation was performed on a reversed phase C18 column using a mobile phase composed of a mixture of 20 mmol/L ammonium acetate buffer solution (pH 4.6) and methanol by isocratic elution. The ratio between mobile phase A and B was 85:15 (V/V) for the detection of benzoic acid and sorbic acid and 95:5 (V/V) for the detection of saccharin sodium and acesulfame K. The analytes were detected using an ultraviolet detector at 254 nm and quantified by an external standard method. The recoveries for benzoic acid, sorbic acid, saccharin sodium and acesulfame K in fermented milk at spiked levels of 1–20 mg/kg were 92.0%–102.4%, and the recoveries in jam at spiked levels of 5–20 mg/kg were 92.3%–105.8%. The limit of quantification (LOQ) for all analytes was 1 and 5 mg/kg in fermented milk and jam, respectively.

A method for the determination of eight colorants, new red, amaranth, allura red, ponceau, tartrazime, sunset yellow, indigotine and brilliant blue in fermented milk by high performance liquid chromatography was developed. The analytes were extracted from samples with weakly alkaline water at 7.7–7.9 and cleaned up on a polyamide solid phase extraction cartridge. The analysis was performed on a C18 column with gradient elution using 0.02 mol/L sodium acetate solution-methanol as mobile phase. The results showed that the recoveries of seven colorants other than indigotine at spiked concentration levels of 0.5–5.0 mg/1 000 g in fermented milk were higher than 90%, while the recovery of indigotine was around 80%. The limit of detection of the method was 0.5 mg/kg. The repeatability relative standard deviation was less than 5%, indicating good precision. With the advantages of accurate results, simplicity and good repeatability, this method is suitable for the analysis of colorants in fermented milk.

A method for determination of hippuric acid and benzoic acid in dairy products by high performance liquid chromatography (HPLC) is described.The protein samples was precipitated by adding lead acetate and centrifuged,and then the analytes were separated on a C18 column with a mobile phase consisting of ammonium acetate and methanol solution and detected with a UV detector at 230 nm.The results showed that hippuric acid and benzoic acid displayed a good linearity relationship in the concentration range of 1-100 mg/kg.The recovery rate was 95.3％-103.6％.The method proved to be simple,accurate and repeatable.

A method for the determination of fructose,glucose,sucrose,maltose and lactose in fermented milk drink by high performance liquid chromatography (HPLC) with refractive index detector was developed.The samples were extracted with water.The method was performed on a carbonhydrate column using acetonitdle-water as mobile phase.The average spiked recoveries of fructose,glucose,sucrose,maltose and lactose spiked in the concentration range of 0.2％-10.0％ were between 92.6％ and 102.0％ with relative standard deviation (RSD,n =6) less than 3.00％.The limit of quantitation was 0.2％.The results indicated that this method is accurate,simple,reproducible and suitable for the analysis of fructose,glucose,sucrose,maltose and lactose in fermented milk drink.

An analytical method to determine lactulose in ultra high temperature (UHT) sterilized milk by liquid chromatographic separation,evaporative light-scattering detection and external standard quantitation is described in this paper.After the protein was precipitated by adding hydrochloric acid,the separation was performed with satisfactory results on a NH2 column using 75％ acetonitrile in water as the mobile phase.The precision expressed as relative standard deviation (n =6) was 0.77％,and the average recovery was in the range of 98.7％-102.6％.This method was simpler and less time consuming with good repeatability than traditional enzymatic hydrolysis method.It can provide an alternative way of determining lactulose in UHT sterilized milk.

A method for the determination of sucralose in dairy products by high performance liquid chromatography (HPLC) was developed.Samples were deproteinized by adding lead acetate followed by cleanup on an Oasis HLB solid-phase extraction cartridge.The separation was performed on a C18 column with 15％ acetonitrile in water as the mobile phase,and the analyte was detected with an evaporative light-scattering detector (ELSD) detector.The recovery of sucralose at spiked concentration levels of 7.5-70 mg/kg was 94.6％-101.3％.The limit of quantitation at a signal-to-noise ratio of 10 was 7.5 mg/kg.This method proved to be accurate,simple and suitable for the analysis of sucralose in dairy products.

A method for the determination of abamectin residues in dairy products and feed by using high performance liquid chromatography (HPLC) with fluorescence detection after precolumn derivation with trifluoroacetic anhydride and N-methyl imidazole was developed.Abamectin residues were extracted with acetonitrile,cleaned up on a tC18 solid phase extraction cartridge.After precolumn derivatization,the separation was performed on C18 column by gradient elution with methano-acetonitrile-water as mobile phase.The analyte were detected with a fluorescence detector and quantified by an external standard method.The recovery of abamectins spiked into milk powder at 1.5 μg/1 000 g (or 1 000 mL for milk) was higher than 90％.The limit of quantitation was 2 μg/kg for dairy products and 10.0 μg/kg for feed.This method proved to be accurate,simple reproducible,and suitable for the analysis of abamectin residues in dairy products and feed.

A method for detecting acesulfame-K, saccharin sodium, benzoic acid, sorbic acid, dehydroacetic acid, methyl vanillin and ethyl vanillin in dairy products by using high performance liquid chromatography (HPLC) is described. The sample was deproteinated and filtered, then separated on a C18 column with a mobile phase made up of methanol and ammonium acetate solution, and detected for absorbance at 230 nm. The seven substances were completely separated within 35 min. Their calibration curves were linear in the range of 1–100 mg/kg with correlation coefficients (R2) all above 0.999 0 under the selected conditions, and the recovery of the proposed method was 80.0% –105.7%. The method proved to be rapid, simple, and reliable.

A method for the determination of lactulose in milk powder by high performance liquid chromatography (HPLC) was developed. Lactulose was extracted with water. The analysis was performed on a NH2 column using 80% acetonitrile in water as the mobile phase. The recovery of lactulose spiked into milk powder in the range of 0.3–2.0 g/100 g was 97.4%– 103.0%. The limit of detection was 0.3 g/100 g. The results indicated that this method is accurate, simple and suitable for the analysis of lactulose in milk powder.

A method for the determination of nucleotides in milk powder by high performance liquid chromatography (HPLC) was developed. Nucleotides were extracted with water, deproteinated with 0.5% acetic acid and cleaned up by an SAX solid phase extraction (SPE) column. The analysis was performed on C18 column using 0.1 mol/L phosphate as the mobile phase. The recoveries of spiked samples at three levels ranging from 0.3 to 50 mg/100 g were higher than 90%. The limit of quantification was 0.3 mg/100 g. This method was accurate, simple, reproducible, and suitable for the analysis of nucleotides in milk powder.

A method for the determination of fructooligosaccharides in modified milk by high performance liquid chromatography (HPLC) was developed. Fructooligosaccharides were extracted with water. The analysis was performed on a NH2 column using 70% acetonitrile in water as the mobile phase. The recoveries of fructooligosaccharides at four spiked levels between 20 and 500 mg/100 g ranged from 80.2% to 107.1%. The limit of quantification was 20 mg/100 g. This method is accurate, simple and suitable for the analysis of fructooligosaccharides in modified milk.

A method for the determination of vomitoxin in milk powder by high performance liquid chromatography (HPLC) was developed. Samples were precipitated and filtered, and the supernatant was then purified and concentrated by immunoaffinity column chromatography, separated by C18 column chromatography, detected by ultraviolet detector and quantified by calibration curve method. The method proved to have good accuracy and precision. The spiked recovery of vomitoxin in dairy products was 98%–106%, and the relative standard deviations were 0.45%–1.21%. The detection limit of vomitoxin was 0.05 mg/kg.

A method for the determination of phosphatidylserine in milk powder by High performance liquid chromatography with evaporative light scattering detection（HPLC-ELSD）was developed. Samples were extracted with a mixture of chloroform and methanol. Chromatographic separation was performed on a Diol column by gradient elution using a binary mobile phase system consisting of hexane- isopropanol-acetic acid triethylamine and isopropanol-water-acetic acid-triethylamine. The recoveries of phosphatidylserine in milk powder at spiked levels between 50 and 150 mg/100 g were greater than 85%, and the limit of quantification was 30 mg/100 g. The proposed method has the advantages of accuracy,