Pan Lijing,Chen Weiwei,Yuan Minjia

Shanghai Qiran Biotechnology Co.,Ltd.,China;

Shanghai Jinjia Technology Co.,Ltd.,China

Abstract An ultrahigh performance liquid chromatography-triple quadrupole tandem mass spectrometry(UPLCMS/ MS) was established to quickly and accurately determine the content of oleuropein in cosmetics.The samples were extracted with methanol-aqueous solution,and the mobile phase with methanol-formic acid solution(0.1 mol/L)=40∶60 was separated by Agilent ZORBAX Eclipse Plus C18 (2.1 mm ×50 mm × 1.8 μm-Micron)column temperature 30 ℃,flow rate 0.3 mL/min.The MS end was detected by electrospray negative modeionization (ESI-) and multiple reaction monitoring (MRM) mode.The results show a good linear relationshipin the range of 0.002～5 mg/L,with a correlation coefficient R2 of 0.999,5.Method recovery range from 84.2%～107.6% and the relative standard deviation RSD is 5.8%.The detection time is 5 min,the detection limitis 0.000,6 mg/L,and the limit of quantification is 0.002 mg/L.This method has the advantages of convenient operation,low quantification limit,high precision and good repeatability,and is suitable for measuring the content of oleuropein in many kinds of cosmetics.

Key words ultra performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS);oleuropein ;cosmetics

Olive is a plant of the genus Melilotus in the family Melilotus,which grows mainly in Mediterranean countries,with the introduction of such species in our country,because of the unique geographical advantages,the olive planting industryin Longnan of Gansu Province and Liangshan of Sichuan Province and other areas has been greatly developed[1,2].The study found that olive leaves are rich in polyphenolic compounds such as oleuropein,hydroxytyrosol and flavones,as the main activeingredient,oleuropein has strong antioxidant activity,antibacterial activity,anti-inflammatory and antibacterial activity,and inhibits the degradation of extracellular matrix in the cornea and the proliferation of epidermal cells to prevent skin from ultraviolet damage[3-6],oleuropein is widely used in skin care products,medicine,food and other fields.

With the wide application of olive leaves,the detection of oleuropein content had been studied more and more.At present,the main detection methods include gas chromatography (GC-MS),UVVIS spectral difference subtraction,high performance liquid chromatography (HPLC-DAD)[7-13].However,the low R value of the calibration curve by GCMS method was easy to cause the deviation of the content result.The high content of oleuropein in the subtraction method can caused serious interference to the results.UPLC-MS/MS has the advantages of high sensitivity,low detection limit,short analysis time,strong selectivity and low false positive probability.However,the detection of oleuropein by UPLC-MS/MS method in the field of cosmetics had rarely been reported,only in the field of biomedicine[14].The UPLC-MS/MS technology was used to determine the content of oleuropein in cosmetics by optimizing pretreatment methods,chromatographic conditions and mass spectrometry conditions.

1 Experimental part

1.1 Instruments and reagents

Agilent 1260 Infinity II-G6465 Liquid mass spectrometer,Agilent Technology Co.,LTD.,ME204E Analytical balance,Mettler Toledo Instrument Shanghai Co.,Ltd.,Millipore-IQ7000 ultra-pure water machine,Merck,Germany,KQ400DE ultrasonic cleaning instrument,Kunshan Shumei Ultrasonic Instrument Co.,Ltd.,IKA-MS 3 Digital Circular shaker,Germany IKA Company,0.22 μm Water membrane filter,Shanghai Anpu Scientific Instrument Co.,Ltd.

Ultrapure water,Millipore-IQ7000;Methanol,HPLC-MS level,Shanghai Anpu Scientific Instrument Co.,LTD;Formic acid,HPLC -MS level;Ethyl alcohol,HPLC level;Acetonitrile,HPLC -MS level,ShanghaiAnpu Scientific Instrument Co.,LTD.,Oleuropein standard(Lot number,2201955),purity 97.6%,ShanghaiAnpu Scientific Instrument Co.,LTD.

Sample information: Olive polyol extract,Draco Natural Products inc;Olive leaf extrac,ShanghaiJiakai Biotechnology Co.,LTD;Total Olea L40,Shanghai Green Die Industrial Co.,LTD;Formulation development in the laboratory: Sample 1,essence;Sample 2,essence;Sample 3,toner;Sample 4,toner;Sample 5,emulsion;Sample 6,emulsion;Sample 7,cream;Sample 8,cream;Sample 9,cream.

1.2 Sample pretreatment

The 0.1 g (accurate to 0.000 1 g) sample was accurately weighed and placed in a 25 mL colorimetric tube,and 50% methanol solution was added to 25 mL at a fixed volume.The sample was extracted by ultrasound for 20 min,and the superwhite was filtered through a 0.22μm Water membrane filter into a brown sample vial for the detection of UPLC-MS/MS.

1.3 Standard solution preparation

Standard stock solution,accurately weigh the oleuropein standard 10 mg (accurate to 0.1 mg).It was dissolved in 50% methanol solution and volume titrated to 25 mL.A standard stock solution with a mass concentration of 400 mg/L was prepared in brown reagent bottles and kept at low temperature and away from light for 1 week.

Remove 1.25 mL of the standard stock solution to a 10 mL volumetric bottle and prepare a 50 mg/Lintermediate solution,then appropriate amount ofintermediate solution was diluted with 50% methanol solution to become 0.002,0.005,0.01,0.5,1,2,5 mg/L standard operating curve,the standard working curve is ready for use.

1.4 Instrument operating condition

1.4.1 Chromatographic condition

Adopt Agilent ZORBAX Eclipse Plus C18 column (2.1×50 mm,1.8-Micron),column temperature of 30 ℃,flow rate was 0.3 mL/min,the sample intake was 2 μL,the mobile phase was methanol -(0.1 mol/L) formic acid aqueous solution(40 ∶ 60),and the elution time was 0～5.0 min.

1.4.2 MS conditions

Using ESI-mode and multiple reaction monitoring scan (MRM),the dryer gas temperatureis 300 ℃,dryer gas flow is 7.0 L/min,atomization gas pressure 35 psi,sheath gas temperature 350 ℃,sheath gas flow rate 11.0 L/min,capillary voltage 4,000 V,nozzle voltage 1,500 V.Qualitative and quantitative monitoring of ion pairs and related parameters are shown in Table 1.

Table 1.Quantitative monitoring of ion pairs and related parameters of oleuropeside

2 Results and discussion

2.1 Selection of the chromatographic mobile phase

According to the chemical structure formula of oleuropein (Table 1),the compound contains polyhydroxyl in the benzene ring and the sugar ring.According to the similar phase dissolution principle,the methanol -(0.1 mol/L) formic acid water system was selected as the mobile phase.Oleuropein was eluted with 20% methanol,40% methanol,60% methanol,80%methanol -(0.1 mol/L) formic acid water respectively,and the oleuropein peak was different (Figure 1).When methanol -(0.1 mol/L) formic acid water (40∶60) was used as the mobile phase,the peak shape of oleuropein was good,the retention time was reasonable,and the sensitivity was high.

Figure 1. UPLC-M S/MS chromatogram of oleuropein at different proportions of mobile

2.2 Investigation of mass spectrum conditions

Oleuropein was analyzed by UPLC-MS/MS,and according to the molecular structure of oleuropein,0.1 mg/L oleuropein standard solution was directly injected for analysis using ESI-mode and multi-reaction monitoring scan (MRM).After optimizd the cracking voltage,collision energy and other parameters,two ion pairs 275.1 and 307.0 were selected for qualitative and quantitative analysis.

2.3 Sample treatment

2.3.1 Selection of the extraction solvent

In the literature,methanol,methanol-water and ethanol-water were commonly used as extractors for oleuropein detection by HPLC-DAD,according to the structural formula,this study carried out proportional optimization on the basis of the literature.The experimental results showed that the peaks of oleuropein were bifurcated and widened when ethanol-water,ethanol and methanol were used as extractants.When methanol-water was used as solvent,oleuropein had a good peak.Under the condition of methanol-water (50 ∶ 50),the peak effect was optimal (Figure 2).Therefore,methanol-water(50 ∶ 50) was selected as the extraction solvent of oleuropein.

Figure 2. UPLC-M S/MS chromatogram of oleurorin at different ratios

2.3.2 Extraction time investigation

The cosmetics with known oleuropein content were extracted by ultrasonic,and the extraction rate of positive samples was investigated when the extraction time was 10,20,30,40 and 60 min respectively.The results showed that when the ultrasonic extraction time was 10 min,the extraction rate of positive sample was 88.0%.When the ultrasonic extraction time was greater than or equal to 20 min,the extraction rate of positive samples reached 95.5%.Finally,ultrasonic extraction time was selected for 20 min.

2.4 Method performance evaluation

2.4.1 Evaluation of matrix effects

Because different substrates may affect the accuracy of the results,this study used different types of blank substrates(toner,emulsion,essence,cream) to add standard products to evaluate the matrix effect (ME) of oleuropein cosmetics.The mass concentration of oleuropein was taken as the horizontal coordinate(X mg/L),the peak area of the quantitative ion pair was the ordinate(Y),to draw solvent standard curve and matrix standard curve,see Figure 3.By calculating the slope ratio of oleuropeinin matrix curve and solvent curve (K matrix/K solvent),the matrix effect of oleuropein was less than 20%,ME=(K matrix/K solvent-1) ×100%,[15-17]indicating that the matrix effect could be ignored and quantified by solvent standard curve.

Figure 3. Standard curves for the different conditions

2.4.2 linear relationship,detection limit and quantitative limit investigation

The standard working solution was prepared.Taking each mass concentration point of oleuropein as the X-axis and its peak response area as the Y-axis,the linear regression equation was obtained:Y=7,7721.36*X－2.259,8,R2=0.999,5.The results indicated that oleuropein had a good linear relationship in the range of 0.002～ 5 mg/L.

The oleuropein standard reserve solution was added to the blank matrix sample,according to the signal to noise ratio (S/N) of 3,the limit of detection(LOD) of oleuropein was 0.000,6 mg/L;when the signal to noise ratio (S/N) of 10,the limit of detection(LOD) of oleuropein was 0.002 mg/L.

2.4.3 Recovery rate and precision

The oleuropein standard solutions of low,medium and high concentrations were precisely added into different blank matrix samples,The relative deviation and recovery rate were tested by parallel experiments for 6 times,and the results were shown in Table 2.The experimental results show that the peak time is stable at 2.5 min.The recoveries ranged from 84.2% to 107.6%,RSD was 5.8%,indicating that the recovery and reproducibility of the method were good and the accuracy was high.

Table 2.Results of oleuropein spike recovery in different matrices (n=6)

2.5 Actual sample test

The commercially available olive extract containing oleuropein and 9 developed formulationsin the laboratory were tested by the experimental method.Parallel experiments were conducted for 3 times,and the experimental results were shownin Table 3.The results show that this method can be applied to practical sample detection.The chromatographic and mass spectrograms of typical positive samples are shown in Figure 4.

Table 3.Results of the determination of oleuropein content in the products

Figure 4. Oliuropeside chromatogram and mass spectrometry of typical positive samples

3 Conclusion

A rapid method for the determination of oleuropein in cosmetics was developed by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS).The sample pretreatment,chromatographic conditions and mass spectrometry conditions were investigated and optimized.Under the optimal conditions,olpicrin could be accurately qualitatively and quantitatively within 5 min.The detection limit,recovery rate and precision could meet the actual test requirements.The method has the characteristics of high sensitivity,short detection time and low quantitative limit,and can be successfully applied to the detection of oleuropein in cosmetics.