José López-Rey Iglesias

Thesis for the degree of European Master in Quality in Analytical Laboratories

Introduction Food additives are substances added to food to preserve

flavor or enhance its taste and appearance.

Introduction

Objective

The aim of the study is to develop a fast, accurate andsensitive HPLC-MS method to simultaneously screen tencommon food additives, quantify them using internalstandard and be able to apply this method innonalcoholic beverages.

Experimental Sample preparation

Experimental HPLC-MS Analysis

- LC 1260 Infiniy

- MS 1100 (ESI source)

- Column Eclipse XDB-C18 (150 x 4.6 mm, 5μm)

Results and discussion Mass spectrum

Compund Most intense signal Fragmentor

TAR 467.00 70

ACS-K 162.00 70

BA 121.00 70

SAC 182.00 70

SA 111.00 70

CYC 178.00 70

NC 537.00 100

SY 407.00 140

IS 192.00 80

SCL 395.00 80

ASP 293.00 80

Table 2. Optimum fragmentor and most intense signal of each food additive

Results and discussion Chromatograms

a) Tartrazineb) Acesulfame-Kc) Benzoic acidd) Saccharin sodiume) Potassium sorbatef) Sodium cyclamateg) Ponceau 4Rh) Sunset yellow FCFi) Internal standardj) Sucralosek) Aspartame

Results and discussion Linearity, LOD and LOQ

Analyte Curve EquationConcentration

range [μg mL-1]R2 LOD LOQ

ACS-K y = -0.0063x2 + 0.4291 + 0.7713 0.1-20 0.9976 0.100 0.300

SAC y = -0.0392x2 + 0.8283x + 0.323 0.1-8 0.9949 0.022 0.066

CYC y = 0.2414x -0.0502 0.1-20 0.9970 0.028 0.085

ASP y = 0.3828x + 0.2444 0.1-20 0.9986 0.061 0.182

SCL y = -0.0006x2 + 0.1158 -0.0031 0.1-20 0.9998 0.056 0.169

BA y = 0.1621x + 0.0294 0.1-20 0.9984 0.055 0.166

SA y = 0.0945x – 0.0019 0.1-20 0.9998 0.038 0.117

TAR y = 0.0007x2 - 0.002x + 0.0027 0.1-8 0.9992 0.049 0.147

NC y = 0.0007x2 + 0.0148x – 0.0135 0.1-8 0.9933 0.019 0.353

SY y = 0.0087x2 + 0.0035x + 0.0036 0.1-8 0.9999 0.013 0.040

Results and discussion Trueness

- Satisfactory recoveries (81.5-108.5%), with relative standard deviations (RSDs) below 6%, were obtained by HPLC-MS for all food additives regardless of the type of sample matrix and the spiking level.

- By HPLC-UV good recoveries (80.3-103.5%) were obtained too, with relative standard deviations (RSDs) below 4%. In general the recovery values are higher when mass spectrometry was used like detector.

Results and discussion Repeatability and reproducibility

- For HPLC-MS the intra-day repeatability was between 1.2-10.7%. The inter-day reproducibility was between 3.3-15.1%.

- For HPLC-UV the intra-day repeatability was between 0.1-2.4%. The inter-day reproducibility was between 0.3-4.7%.

Results and discussion Analysis in real samples: sweeteners

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ACS-K

SAC

CYC

SCL

ASP

Results and discussion Analysis in real samples: preservatives

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SB

PS

Conclusion A method for simultaneous determination of ten food

additives (ACS-K, ASP, BA, CYC, NC, SA, SAC, SCL, SY and TAR) by HPLC-MS has been described. Application of mass spectrometry allowed the determination of the ten compounds with high sensitivity and selectivity in a single analysis. The quantitation of the analytes was carried out with the help of an internal standard, N-(2-methylcyclohexil)sulfamic acid, avoiding possible influences from mass spectrometry detector or changes in injection volume. The sample preparation is easy and fast.

The proposed method has been successfully applied for determination of food additives in real samples.