Hittite Journal of Science and Engineering, 2016, 3 (2) 91-97 ISSN NUMBER: 2149-2123 DOI: 10.17350/HJSE19030000037
Validation Rf H3/& Method Ior Whe Determination RIK\GUR[\PHWK\OIXUIXUDOLQ3HVWLO.|PH-DP0DUPDODGH$QG 3HNPH]
&HPDOHWWLQ%DOWDFÕDQG=H\QHS$NúLW Gumushane University, Department of Food Engineering, Gumushane, TURKEY
Article History: ABSTRACT Received: 201͞/1͘/1͝ Accepted: 2016/͙͚/͚͙ Online: 2016/͙͚/3͙ his study represents a high performance liquid chromatography (HPLC) method for the detection of 5-hydroxymethylfurfural in pestil, köme, jam and pekmez T Correspondence to: Ǥ Çǡ samples. The linearity, selectivity, decision limit, detection capability, detection limit, ǡ quantification limit, precision, recovery, ruggedness and measurement uncertainty of ǡ͚͙͘͘͡ ǡ the method were determined. The developed method, simple and accurate, showed ǣή͘͡ȋ͜͝͞Ȍ͚͙͚͙͛͛͡Ȁ͚͚͛͛ good recovery values (97-108%). The accuracy of the method expressed with the relative ǣή͘͡ȋ͜͝͞Ȍ͚͙͚͚͛͛͡ standard deviation was below 6%. The detection limit and quantification limit were 0.03 E-Mail: [email protected] mg/kg and 0.10 mg/kg, respectively. HMF levels in pestil, köme, jam, marmalade and pekmez samples were determined using the validated method.
Keywords: HMF; Method validation; Pestil; Köme; Jam; Marmalade and Pekmez.
INTRODUCTION
-Hydroxymethylfurfurol (HMF) is a furanic determination of HMF content can be used to evaluate 5compound formed under acidic conditions by the the effects of food processing industry and storage Maillard reaction or sugar dehydration [1]. Maillard conditions on the quality of food products [7, 2]. HMF reaction is a non-enzymatic browning reaction, has been used to evaluate the sensorial properties of occurs when foods including reducing sugars and food products. The changes in the color, flavor and taste amino acids are heated. HMF is an intermediate of food products during processing and storage are product of this reaction. Moreover, HMF formation related to the HMF content. Hence it is recognized as an takes place during hexoses dehydration at lower pH indicator of improper processing and storage conditions (< 5) via enolisation, for which the presence of amino [8, 9]. The Turkish Standards [10, 11] state a maximum acid groups is not needed [2]. HMF level of 50 mg/kg in pestil and köme and jam [12]. The established maximum HMF levels are 75 mg/kg, HMF and its derivatives have been reported to and 100 mg/kg for liquid pekmez, and solid pekmez in show toxic properties such as cytotoxic, genotoxic, accordance with [13]. nephrotoxic, mutagenic and cancerogenic. The presence of HMF in foods has gained interest due to the In the past, a great number of the methods toxicological concerns about HMF. Although further developed for the detection of HMF in foodstuffs studies have revealed that HMF does not exhibit a were based on spectrophotometric techniques [14, 15]. crucial health risk, it has been a matter of debate [3]. Although spectrophotometric methods are fast, their sensitivity and specificity are low. Chromatographic Although HMF is nearly absent in untreated foods, methods have been used and developed to detect it occurs in processed foods containing carbohydrates HMF compounds in food products. UV detection of such as bread, biscuits, jam, marmalade, honey and High performance liquid chromatography (HPLC) fruit juice [4, 5, 6]. HMF amount tends to increase is mostly used method used for the determination of during heat treatment and storage. Therefore, the HMF in foodstuffs. Accuracy and sensitivity of the Ǥ Ç ǤçǤȀ ǡ͜͢͞͝ǡ͟ȋ͞Ȍͥ͝–ͥͣ Analytical chemicals and HPLC grade solvents were solvents grade HPLC and chemicals Analytical The HPLC method based the method for HMF detection detection for HMF method the based method HPLC The P (90:10 v/v), was at a flow rate of 1 mL/min, wavelength at wavelength (90:10 of1mL/min, rate v/v), at aflow was (IKA, Germany). 5 g of the sample was dissolved with 25 25 with dissolved was sample 5gofthe Germany). (IKA, (MS) analysis has been proposed for HMF determination determination for HMF proposed been has (MS) analysis 285 nm. 285 METHODS Millipore (Bedford, MA, USA). MA, (Bedford, Millipore (45 MO, filters USA). Membrane Chemicals Method validation UV system (Agilent 1100 series, USA). The separation of USA). separation The 1100 series, (Agilent system UV HLPC- an out using carried was analysis Quantitative were collected samples pekmez and köme, jam Pestil, Specimens were homogenized by an Ultra Turrax mixer mixer Turrax Ultra byan were homogenized Specimens Practising Equipment obtained from Merck (Darmstadt, Germany). HMF HMF Germany). (Darmstadt, Merck from obtained carried out as well. well. out as carried different matrices because of the potential toxic effects of of effects toxic potential ofthe because matrices different sensitive Reliable, samples. honey for primarily developed Samples Sample preparation Sample was added to the mark. The sample solutions were solutions sample The mark. tothe added was Isolution ofCarrez mL 0.5 flask, mL a100 in water mL methods. Gas chromatography (GC)-mass spectrometry spectrometry (GC)-mass chromatography Gas methods. HMF was carried onaC carried was HMF HPLC method are better than that of spectrophotometric HMF and quality control of food products. The goal of this ofthis goal The products. offood control quality and HMF respect to decision limit, detection limit, quantification limit, filtered through 45 45 through filtered from a local market in Trabzon and Gümüşhane. All All Gümüşhane. Trabzon and in market alocal from jam and pekmez. The proposed method was validated with with validated was method proposed The pekmez. and jam and 0.5 mL of Carrez II solution were added, later water water later were added, solution II ofCarrez mL 0.5 and as well [5]. standard (99%) was bought from Sigma-Aldrich (St. Lois, (St. Lois, Sigma-Aldrich from bought (99%) was standard and rapid method for the detection of HMF in pestil, köme, köme, pestil, in ofHMF detection for the method rapid and in HMF todetermine required are methods rapid and sample solution was injected to the HPLC-UV system. HPLC-UV tothe injected was solution sample samples were stored at 4 were stored samples selectivity, linearity, precision, recovery and ruggedness. ruggedness. and recovery precision, linearity, selectivity, in honey samples was validated and applied to pestil, köme, topestil, applied and validated was samples honey in investigation was to develop and validate a sensitive, reliable reliable asensitive, validate and todevelop was investigation m (Nucleosil, USA). The mobile phase, water-methanol water-methanol phase, USA). mobile The m (Nucleosil, The methods for the determination of HMF have been been have ofHMF determination for the methods The of the developed procedure to real samples was was samples toreal procedure developed ofthe P m membrane filters. 100 100 filters. m membrane q C until analysis. C until 18 column, 250 mm 250 column, P m) were supplied by u 4.6 mm, 5 mm, 4.6 P L of the L ofthe 92 A calibration curve was obtained by plotting the peak peak the byplotting obtained was curve A calibration Validation The Limit of quantification ( ofquantification Limit The todetect. ability the describes ofamethod selectivity The Microsoft Excel 2007 (Microsoft Corp.,Redmond, WA, Corp.,Redmond, (Microsoft 2007 Excel Microsoft RESULTS AND DISCUSSION RESULTS AND Linear regression model was performed using the least least the using performed was model regression Linear USA) was used for data processing. Outliers were checked were checked Outliers processing. for data used USA) was Single laboratory validation was performed according to according performed was validation laboratory Single value higher than 0.999 (Table 1). Preparation and mass (Table 0.999 mass and 1). than Preparation higher value Limit of detection and limit ofquantification limit and ofdetection Limit Linearity quantification limit, precision, recovery, ruggedness and and ruggedness recovery, precision, limit, quantification concentration of calibration used for HPLC-UV and and for HPLC-UV used ofcalibration concentration equation described as curve calibration The concentrations. different of five of selectivity the process, validation the beginning Before decision limit, detection capability, detection limit, limit, detection capability, detection limit, decision obscure. were selectivity, determined method of validated Statistical analysis Selectivity method should be checked against naturally occurring occurring naturally against checked be should method measurement obscure of the method were ascertained were ascertained method ofthe obscure measurement Regulation 2004/882/EC. Performance characteristics Honey Commission Honey from Fig. 1, no interference observed at the retention jam, marmalade marmalade jam, area of standard solution in terms of absorbance, with were compared chromatograms their and analyzed and removed based on the Cochran test and Grubbs test. test. Grubbs and test Cochran onthe based removed and areas of standard solutions which were the three series series three were the which solutions ofstandard areas selective for HMF analysis. analysis. for HMF selective squares approach. squares some analytical parameters from the developed method. method. developed the from parameters analytical some ( samples blank Representative substances. samples. linearity was obtained in the studied range, with R with range, studied the in obtained was linearity the concentration of standard solution in mg/kg. Good was method proposed the that indicated ofHMF time seen be can As samples. spiked ofthe chromatogram the statistical certainty. If both accuracy and precision are are precision and accuracy both If certainty. statistical to validate the method for HMF analysis in the studied studied the in analysis for HMF method the to validate limit, precision, recovery, ruggedness and measurement measurement and ruggedness recovery, precision, limit, decision limits, quantification and detection linearity, the analyte which can be measured with reasonable reasonable with measured be can which analyte the and pekmez samples of samples pekmez and [16]. The sensitivity, linearity, [16]. linearity, sensitivity, The y =a( x ) + b, where ) +b, where LOQ), lowest content of lowest LOQ), the International International the y is the peak peak the is n=20 ) were x is 2 Table 1. Preparation and mass concentration of calibration used for HPLC-UV and some analytical parameters
Main stock ȋσȌ b͚͝ solution ȋ-1Ȍ as calibrator ȋ-1Ȍ
1 st 2 nd 3 nd 4 th 5 th 1 st 2 nd 3 nd 4 th 5 th 1000 a 25 50 100 200 300 1.0 2.0 4.0 8.0 12.0
ȋȀȌ 1.0-12.0
Calibration equation ycγ͛͘͟͞Ȃ͘Ǥ͛
ƥ ͘Ǥ͡͡͡
a HMF standard was used 99% purified b Dilution of V i of the stock solution to 25 mL with purified water, to produce calibrations i c y is the peak area of standard solution expressed in absorbance, x is the concentration of standard solution expressed in mg/kg.
Figure 1. A) representative blank sample B) spiked specimen constant over a concentration rangearound the limit of standard deviation of the mean of 0.2 mg/kg. LOD and detection, then the limit of quantification is numerically LOQ were determined by analyzing ten samples spiked equal to 10 times the standard deviation of the mean of with HMF (0.2 mg/kg) in accordance with Analytical 0.2 mg/kg. The limit of detection (LOD), expressed as Detection Limit Guidance [17]. In order to estimation the concentration, or the quantity, is derived from the of LOD, the standard deviation of the response (s) was smallest measure, that can be detected with reasonable multiplied by the Student’s t-test value for ten replicates certainty for a given analytical procedure. the limit and nine degrees of freedom. The forecasted LOD values of quantification is numerically equal to 3 times the were confirmed according to the guidance [17] as well.
Table 2. The decision limit (CCα) and detection capability (CCβ) values of the method calculated at the MRL (n=20) for the HMF Ȁ ǡ͜͢͞͝ǡ͟ȋ͞Ȍͥ͝Ȃͥͣ Added Measured ± S.D. Ƚ Added Measured ± S.D. Ⱦ Matrix CC ȋȀȌ CC ȋȀȌ ȋȀȌ ȋȀȌ ȋ͙Ǥ͜͞ǤǤȌ Ƚ ȋȀȌ ȋȀȌ ȋ͙Ǥ͜͞ǤǤȌ Ⱦ 0.03a 0.04 ± 0.02 0.03 0.06 0.06 0.05 ± 0.01 0.02 0.08 Jam 50.00b 50.50 ± 0.88 1.44 51.44 51.44 50.65 ± 0.62 1.02 52.46 0.03a 0.03 ±0.01 0.02 0.05 0.05 0.04 ±0.01 0.02 0.08 pekmez ͟͝Ǥ͘͘b ͟͜Ǥ͘͝ΰ͘Ǥ͞͞ 1.08 ͟͞Ǥ͘͠ ͟͞Ǥ͘͠ ͟͝Ǥ͘͝ΰ͘Ǥ͟͡ 1.30 ͟͟Ǥ͛͠ Solid 0.03a 0.04 ± 0.02 0.03 0.06 0.06 0.04 ± 0.02 0.03 ͘Ǥ͘͡ pekmez 100.00b ͡͡Ǥ͘͝ΰ͙Ǥ͘͞ ͙Ǥ͟͜ ͙͙͘Ǥ͟͜ ͙͙͘Ǥ͟͜ ͙͘͘Ǥ͘͝ΰ͘Ǥ͡͠ 1.61 103.35 0.03a 0.028 ± 0.01 0.02 0.05 0.05 0.048 ± 0.01 0.02 ͘Ǥ͘͟
Ǥ Ç ǤçǤ 50.00b ͜͡Ǥ͘͠ΰ͘Ǥ͟͜ 1.21 51.21 51.21 ͘͝Ǥ͘͠ΰ͘Ǥ͟͞ 1.10 52.31 0.03a 0.028 ± 0.01 0.02 0.05 0.05 0.045 ± 0.02 0.10 ͘Ǥ͘͟ Pestil 50.00b ͜͡Ǥ͙ΰ͘Ǥ͙͡ ͙Ǥ͜͡ ͙͝Ǥ͜͡ ͙͝Ǥ͜͡ ͙͝Ǥ͚͘ΰ͘Ǥ͚͟ 1.18 ͚͝Ǥ͟͞ a limit of quantification, b maximum permitted limit
93 Ǥ Ç ǤçǤȀ ǡ͜͢͞͝ǡ͟ȋ͞Ȍͥ͝–ͥͣ Two new performance characteristic of decision limit limit ofdecision characteristic Two performance new (CC a 20 blank samples fortified with HMF at the maximum maximum at the HMF with fortified samples blank 20 %). CC The Decision limit and detection capability detection and limit Decision LOQ values were evaluated as ten times of the standard standard ofthe times ten as wereevaluated values LOQ deviation. deviation. Table 3. Table 3. Precision was determined by analyzing twelve empty empty twelve byanalyzing determined was Precision CC calculated in Table in 2. calculated deviation. The determined LOD and LOQ values for values LOQ and LOD determined The deviation. detected, identified and/or quantified in a sample (β=5 asample in and/or quantified identified detected, deviation of the fortified samples [1] samples CC fortified ofthe deviation Precision Regulation 2002/657/EC. CC 2002/657/EC. Regulation HMF substances were 0.01 mg/kg and 0.03 mg/kg, mg/kg, 0.03 and were 0.01 mg/kg substances HMF respectively. respectively. permitted limit (MRL) regulated by Turkish Food Codex Codex Food byTurkish regulated (MRL) limit permitted according to the following equations of the method were method ofthe equations following tothe according and Turkish Standards. Estimated CCα and CC and CCα Estimated Standards. Turkish and standard deviation of the fortified samples [2] samples fortified ofthe deviation standard specimen spiked with HMF standard solution at the standard HMF with spiked specimen limit of samples concluded as non-compliant(α =5%) and non-compliant(α as concluded ofsamples limit 0.5x MRL, Jam Analyte pekmez Solid Pekmez Pestil β α refers the lowest content of the substances that may may that substances content ofthe lowest the refers CC ) and detection capability (CC capability detection ) and b The repeatability and within-laboratory reproducibility of the method, expressed with the standard deviation and the relative s relative the and deviation standard the with expressed method, the of reproducibility within-laboratory and repeatability The MRL, α = the concentration at MRL+ 1.64 1.64 at MRL+ concentration = the 25.00 25.00 25.00 50.00 50.00 50.00 50.00 ͛͟ǡ͘͝ 150.00 100.00 112.50 ͟͝Ǥ͘͘ ͟͝Ǥ͘͘ ͟͝Ǥ͘͘ ͟͝Ǥ͘͘ mg/kg Ƥ level c α 1.5xMRL and CC a c c b c b b a b a a a c c b β values were determined to analyze ͜͠Ǥ͡͞ ͜͠Ǥ͛͡ ͜͡Ǥ͛͞ ͚͝Ǥ͡͞ 24.02 25.42 51.23 ͡͡Ǥ͚͜ ͛͞Ǥ͜͡ ͙͙͙Ǥ͟͡ 148.30 ͟͜Ǥ͘͠ ͟͜Ǥ͙͝ ͟͜Ǥ͚͠ ͟͜Ǥ͛͝ mg/kg Determined level ǦȋγȌ Ǧȋγ͞Ȍ Ǧȋγ͞Ȍ α refers to the above the the above tothe refers 1.08 ͙Ǥ͙͡ 1.26 ͙Ǥ͛͡ 1.58 ͙Ǥ͘͟ 1.58 ͙Ǥ͘͟ 1.24 ͙Ǥ͞͡ 1.05 0.88 ͘Ǥ͟͡ ͘Ǥ͛͡ ͘Ǥ͛͡ β mg/kg S ), are submitted by submitted ), are r β = CC x the standard α + 1.64 + 1.64 4.11 2.13 2.08 2.11 2.18 2.80 3.22 3.58 ͛Ǥ͟͠ 3.45 1.18 ͙Ǥ͚͟ ͙Ǥ͟͞ ͘Ǥ͟͡ 0.65 ȋάȌ RSD Precision β values r x the 94 ͜Ǥ͟͡ ͝Ǥ͙͡ 5.51 5.51 5.86 5.51 5.86 5.51 5.86 5.28 5.86 0.66 X ȋάȌ 6.12 6.51 6.51 6.51 Horwitz 75, and 112.5 mg/kg for liquid pekmez) were analyzed were analyzed pekmez) for liquid mg/kg 112.5 75, and Recovery values at the three concentration levels were found to be tobe were found levels concentration three at the values concentrations of the HMF standard were added to were added standard HMF ofthe concentrations since oftrueness instead determined was method of the obtained ranged from 97.18% to 107.68% (Table 4), operators in three days over a month. The results for the for the results The overamonth. days three in operators precision, one day. intermediate the To determine For the MRL. times 1.5 1and of0.5, concentrations deviation (RSD deviation Recovery is a measure of the accuracy. The The accuracy. of the ameasure is Recovery reference material cannot be available. Three different different Three available. be cannot material reference repeatability expressed with the standard deviation (S deviation standard the with expressed repeatability precision test, specimens were conducted in ten replicates replicates ten in were conducted specimens test, precision for jam; 50, 100, and 150 mg/kg for solid pekmez; 37.5, pekmez; for solid mg/kg 150 and 100, 50, for jam; for the within-laboratory reproducibility expressed with with expressed reproducibility within-laboratory for the and the relative standard deviation (RSD deviation standard relative the and kg for köme, jam and pestil; 37.5, 75, and 112.5 mg/kg 37.5, mg/kg 112.5 75, and pestil; and kg for köme, jam and analyses were fulfilled by the same operator in in operator same bythe were fulfilled analyses and samples, 0.5, 1 and 1.5 times MRL. (25, 50, and 75mg/ and (25, 50, MRL. times 1.5 1and 0.5, samples, samples (ten replicates) were analyzed by two different different bytwo (ten were analyzed replicates) samples the Horwitz equation. equation. Horwitz the from (Table 3) values reference calculated the lower than to determine the recovery values. The recovery values values recovery The values. recovery the to determine the standard deviation (S deviation standard the
͜͠Ǥ͟͠ ͜͡Ǥ͛͟ ͜͡Ǥ͠͞ ͚͛Ǥ͝͡ 24.56 24.54 50.80 ͛͟Ǥ͙͡ ͡͡Ǥ͜͠ 112.24 ͙͜͡Ǥ͠͝ ͟͜Ǥ͟͡ ͛͟Ǥ͘͡ ͟͜Ǥ͜͡ ͟͜Ǥ͚͛ mg/kg Determined level R ) are presented in Table 3. Both RSD Table RSD in Both 3. presented ) are ͙Ǥ͞͡ 1.13 1.22 ͙Ǥ͚͟ 1.18 ͙Ǥ͙͟ ͙Ǥ͠͡ 1.25 1.21 ͙Ǥ͠͡ 1.25 1.21 1.15 ͙Ǥ͙͟ 1.56 mg/kg S R R ) and the relative standard standard relative the ) and ͜Ǥ͛͡ 2.35 2.52 ͚Ǥ͟͝ 2.35 2.56 2.46 5.13 3.22 1.50 1.51 1.15 1.31 1.55 ȋάȌ 6.36 RSD Precision R
r ) and the results results the ) and reclamation reclamation ͡Ǥ͚͟ ͡Ǥ͠͞ ͡Ǥ͠͞ ͡Ǥ͠͞ ͟Ǥ͠͞ ͟Ǥ͛͝ ȋάȌ 8.35 8.00 8.88 8.35 8.88 8.35 8.88 8.35 8.88 Horwitz tandard r ) Table 4. The recovery values of spiked samples at three different concentrations (n=6).
Mean Determined Mean Recovery RSD Sample Spiked level (mg/kg) S (mg/kg) Level (mg/kg) (%) (%)
Jam 25.00a 24.60 98.40 0.44 1.79
50.00b 49.11 98.22 0.58 1.18
75.00c 73.62 98.16 1.05 1.43
Liquid Pekmez 37.50a 37.14 98.80 0.46 1.24
75.00b 76.33 99.04 0.53 0.69
112.50c 110.65 98.36 1.47 1.33
Solid Pekmez 50.00a 49.57 99.14 0.68 1.37
100.00b 99.69 99.69 0.96 0.96
150.00c 148.44 98.96 0.85 0.86
Kome 25.00a 26.92 107.68 0.64 2.38
50.00b 49.53 99.06 1.52 3.06
75.00c 74.12 100.2 0.93 1.25
Pestil 25.00a 23.92 98.83 0.64 2.68
50.00b 48.59 97.18 1.52 3.13
75.00c 74.54 99.39 0.93 1.25 a0.5x MRL, bMRL, c1.5xMRL showing good recovery values for the proposed method. against the changes in the procedure. As a result, the proposed useful way was validated for the determination of Ruggedness HMF in pestil, köme, jam and pekmez. Ruggedness of the method was evaluated by Youden test. Eight experiments were performed to evaluate the seven Measurement uncertainty selected factors: extraction solution, specimen matrix, The validation data was used to calculate the specimen preparation, analyst, column temperature, measurement uncertainty [19]. Volume, mass, HPLC column and mobile phase. The standard deviation calibration curve, reproducibility and repeatability of of impacts was evaluated according the following the method, preparation of standard, accuracy and equation. reproducibility of the equipment were selected as sources of uncertainty budget. The relative expanded uncertainty [3] of measurement was reckoned using a coverage factor k=2, corresponding approximately 95% confidence level. Expanded uncertainty value was 9.1% for HMF in E n Where i is each of the calculated effect, and is the foodstuffs. number of parameters. Application of the method to real samples The influence of the factors on method performance Pestil, köme, jam, and pekmez samples were analyzed t has to be checked applying the -test [18]. The experimental using the validated method. Pekmez, a traditional food t Ȁ ǡ͜͢͞͝ǡ͟ȋ͞Ȍͥ͝Ȃͥͣ -values for the factors were calculated according to the product in Turkey, is concentrated grape or mulberry formula given below: juice formed by boiling without the addition of sugar and another ingredients [20]. Pestil and köme in Turkey [4] are made from both fruit juice and concentrated fruit juice [21]. The HMF content of pekmez samples (solid n:25 and liquid) was found to be ranged from 1.44
The experimental t-values (Table 5) were found to be to 66.30 mg/kg, complying with the values set by the Ǥ Ç ǤçǤ t lower than the critical value ( crit = 2.45 at 95% confidence Turkish Codex [13]. Available data on the HMF content level), indicating that the method is sufficiently rugged of pekmez is limited. Our results seemed to be higher
95 Ǥ Ç ǤçǤȀ ǡ͜͢͞͝ǡ͟ȋ͞Ȍͥ͝–ͥͣ An method for the extraction of 5-HMF from food food from of5-HMF extraction for the method An These values were in agreement with data found in the the in found data with agreement were in values These 1.
Table 5. Table 2004/882/EC. The validated method provides accurate accurate provides method validated The 2004/882/EC. CONCLUSIONS liquid conclusion can be derived that recommended method is is method recommended that derived be can conclusion compared to the literature[22]. The HMF content of jam content ofjam HMF literature[22]. The tothe compared with UV detection was validated according to Regulation toRegulation according validated was detection UV with were in agreement with data found in the literature [2, literature the in found data with agreement were in results and offers quick and economic procedure. The The procedure. economic and quick offers and results 8]. The HMF content of Pestil and köme (solid and content ofPestil 8]. HMF The 2. samples and its subsequent determination using HPLC HPLC using determination subsequent its and samples such as köme, pestil, jam, and pekmez. pekmez. and jam, köme, pestil, as such matrices food the in ofHMF detection for the suitable samples ( samples literature [23]. a Originalparameters, a- iE*it Ei*Ei Ei 8 7 6 5 4 3 2 1 -b +a 934. 924. 4. 4. 4. 5. 03001200.21 0.12250 -0.350 -51.2 -48.9 -48.2 -48.1 47.9 49.2 48.6 v/v) (90/10 /Methanol Water a. 49.3 a A Water solvent Extraction A. 93-86-924. 4. 824. 5. 07004000.86 0.49000 -0.700 -51.2 48.9 48.2 -48.1 47.9 -49.2 -48.6 49.3 g G 93-864. 4. 81-824. 5. 01000000.02 0.01000 -0.100 -51.2 48.9 -48.2 48.1 ) v/v (80/20 Water/methanol g. -47.9 49.2 -48.6 49.3 c II Iand carrez of addition No c. ) v/v (90/10 methanol Water/ C phase Mobile G. II Iand carrez of Addition preparation Sample C. 93-86-924. 81-82-895. .0 .28 0.04 0.02286 0.400 51.2 -48.9 -48.2 48.1 47.9 -49.2 -48.6 49.3 f F 93-864. 4. 4. 82-895. .0 .10 2.12 1.21000 1.100 51.2 -48.9 48.2 -48.1 -47.9 49.2 -48.6 49.3 e E 934. 4. 4. 4. 4. 895. .5 .25 2.32 1.32250 1.150 51.2 48.9 -48.2 -48.1 -47.9 -49.2 48.6 49.3 d D D. Analyst Analysts 1 d. Analysts 2 Analysts d. 0C 27 e. C18 5mm mm´4.6 250 mm, 1 Analysts temperature) (Room 0C 22 F. LCcolumn temperature Column E. D. Analyst 934. 4. 4. 814. 4. 5. 07005200.98 0.56250 -0.750 -51.2 -48.9 48.2 48.1 -47.9 -49.2 48.6 49.3 b sample Blank b. B pekmez and marmalade jam, köme, Pestil, matrix Sample B. atrOiia aaees(-)Changed parameter (a-g) Original parameters (A-G) Factor 126, Pages 1192, 2011. Pages 126, Teixido, E., Nunez, O., Santos, FJ., Galceren, MT. FJ., O., Galceren, Santos, Nunez, E., Teixido, Capuano,E. And Fogliano,V. 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