A validated liquid chromatographic method for the determination (1)

A validated liquid chromatographic method for the determination of polycyclic aromatic hydrocarbons in honey after homogeneous liquid-liquid extraction using hydrophilic acetonitrile and sodium chloride as mass separating agent.

PRESENTED BY: i. AHMED MATEEN

ii. FAREEHA SIDDIQI

Anastasia Koltsakidou, Konstantinos Fytianosa Environmental Pollution Control Laboratory, Chemistry Department, Aristotle University of Thessaloniki, Thessaloniki 54124, GreeceConstantinos K. ZacharisResearch Laboratory for the Physical and Chemical Testing of Foods, Department of Food Technology, School of Food Technology and Nutrition, Alexander Technological Educational Institute (ATEI) of Thessaloniki, 57400, Thessaloniki, GreeceAnalytical Development Laboratory, R&D API Operations, Pharmathen SA, Building Thermi 1, 9thklm Thessaloniki-Thermi, Thessaloniki, 57001, Greece

ABSTRACT

GOOD NEWS

• In the present report, a simple and cost-effective method for the determination of twelve US EPA priority polycyclic aromatic hydrocarbons (PAHs) in honey samples after salting-assisted liquid–liquid extraction and UHPLC with fluoresc.ence detection

SAMPLE TREATMENT:

Hydrophilic Acetonitrile (ACN ) as extraction solvent and its phase separation under high salinity conditions.

Phase separation due to the high sugar content of the samples .

Analysis of the selected PAHs residues in various honey samples obtained from Greek region.

PARAMETERS EFFECTING EXTRACTION EFFICIENCY & METHOD SENSITIVITY:

i. Concentration of the honey samples.ii. Type and volume of the extraction solvent.iii. Type and quantity of the inorganic salt. iv. Extraction time .v. Centrifugation time .

The limit of detection (LOD) of the method lay between 0.02 and 0.04 ng/ mL(corresponding to 0.08 and 0.16 ng/g).

The mean analytical bias (expressed as relative recoveries) in all spiking levels was acceptable being in the range of 54–118% while the relative standard deviation (RSD) was lower than 19%.

INTRODUCTION

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Polycyclic Aromatic Hydrocarbons‡ (PAHs)

• Group of more than 100 different chemicals containing 3 or more fused aromatic rings:

E.g.,

anthracene naphthacene

• Health hazard – many PAHs are known carcinogens

• Formed mainly as a result of incomplete combustion – widespread and strongly associated with human activity

• Associate with particulate matter, soils, and sediments

Also known as polyaromatic hydrocarbons or polynuclear aromatic hydrocarbons

coronene

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SOURCES OF PAH PAHs are predominantly anthropogenic and are

formed by: Incomplete combustion of organic matter such as

coal, wood, oil, petrol and diesel. Coke and Al production, bitumen production,

vehicle and aircraft exhaust. Smoking cigarettes. Charbroiled meats.

PAHs are also found in natural fuel deposits.

A few PAHs are used to produce medicine, dyes, plastics, & pesticides

Natural sources of PAHs include volcanoes and natural fires

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PAHs can be found in water also as a direct pollution from industries or from road runoff.

They can settle in the sediment, remain in the water or be taken up by organisms like plankton, mollusks and fish, thereby entering the food chain.

E.g., In the USA, residential wood and coal combustion produces about 700 tons/yr of PAHs compared to 1 ton/yr by coal power stations

Source%

Heating, power production51

Industrial producers20

Incineration & open burning28

Vehicles 1

B(a)P in foodstuffs μg/kgCharcoal broiled steak 8Margarine 1-

36Sausages 4-

50Roasted coffee 1-13Toast 0.5

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16 EPA “priority PAH pollutants” Naphthalen

eAcenaphtheneAcenaphthylene Fluorene

Phenanthrene Anthracene FluoranthenePyrene

Benzo(a)anthracene Crysene

Indeno(1,2,3-cd)pyrene

Benzo(k)fluoranthene

Benzo(a)pyreneDibenzo(a,h)anthracene Benzo(ghi)perylene

Benzo(b)fluoranthene

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PAH4

Benzo(a)anthracene Crysene

Benzo(a)pyrene Benzo(b)fluoranthene

13

PAH8Benzo(a)anthracene

Crysene

Benzo(a)pyrene

Benzo(b)fluoranthene

Benzo(k)fluoranthene

Dibenzo(a,h)anthracene

Indeno(1,2,3-cd)pyrene

Benzo(ghi)perylene

Scientific panel of Contaminants in the Food Chain (CONTAM panel) of EFSA concluded that PAH4 & PAH8 are currently the most appropriate indicators of the carcinogenic potency and effect of PAHs in foodstuffs

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11 PHA Studied Fluorene Phenanthrene Anthracene Pyrene

Benzo(a)anthraceneChrysene

Indeno(1,2,3-cd)pyrene

Benzo(k)fluoranthene

Benzo(a)pyreneDibenzo(a,h)anthraceneBenzo(ghi)perylene

Benzo(b)fluoranthene+

CONVENTIONAL TECHNIQUES FOR PAH DETERMINATION IN FOOD

PRODUCTSThe analysis of PAHs in honey samples is mostly performed by conventional sample pretreatment techniques such as, Liquid-liquid extraction (LLE). Solid-phase extraction. (SPE).

For separation of analytes with separation techniques(GC or HPLC) coupled to optical (fluorescence) or mass spectrometric detectors are used frequently.

DISADVANTAGES

LLE is generally laborious and time-consuming procedure while large volumes of organic toxic solvents are usually required (e.g. CH2Cl2).

SPE is considered as the technique of choice in many laboratories, it requires additional hardware and consumables increasing the entire operational costs.

So,Their was a need for cost effective and

rapid analytical technique for Determination

Introduced by Matkovich and Christian. Extract metal chelates using a binary homogenous

system of acetone and water. In this technique a low dielectric water miscible

organic solvent such as methanol acetone and acetonitrile is used. A little amount of inorganic salts is also added.

The addition of an inorganic salt into a mixture of water and a water-miscible organic solvent causes a separation of the solvent from the mixture and the formation of a two-phase system.

SALTING OUT ASSISTED LIQUID LIQUID EXTRACTION

SALTING OUT PHENOMENON:i. More polar solvent favorably surrounds the electrolyte

(salt) due to the Coulombic forces. ii. One of the solvents preferentially solvates the electrolyte

(ionic or not ionic) making it unavailable to dissolve the other solvent).

It is possible the above phenomena can act synergistically.

FACTORS ON WHICH SALLE: iii. The solvent type and volume iv. Salt type and amountv. Sample concentration which are needed to be examined

and optimized in order to establish the optimum working conditions.

ADVANTAGES.

Operation simplicity. Low cost. Reduction of Extraction time. No use of Organic/aromatic solvents. Fast Mass transfer due to infinite contact area b/w the

two phases. Vigorous Shaking and sonication usually not required. Similarly pH and temperature have less effect on

separation/extraction as compare to liquid-liquid extraction or Dispersive liquid-liquid micro extraction (DLLME).

EXPERIMENTAL

REAGENTS: A certified mixture of 13 PAHs (EPA 525 PAH Mix A)

containing 500 µg /mL each in dichloromethane was provided by Supelco Analytical.

Acetonitrile (ACN), acetone, ethanol and isopropanol were of HPLC grade and supplied by Panreac (Barcelona, Spain).

Methanol was purchased by Fluka. Salts including MgSO4, Na2SO4, (NH4)2SO4,

ZnSO4were provided by(Merck, Darmstadt, Germany). NaCl from Chem-Lab NV (Zedelgem, Belgium). Ultrapure water (18 MΩcm resistivity) was provided by

Millipore Direct-Q UV, Millipore S.A.S., (Molsheim, France).

SOLUTION & WORKING STANDARD:

A standard PAHs stock mixture (50 mg /L) was prepared in ACN stored at −18C and protected from the light. Working standard solutions were prepared daily in HPLC grade Acetonitrile.

INSTRUMENTATION

Acquity UPLC binary solvent system (Waters) with a fluorescence and/or PDA detector.

Analytical column was a reversed phase Acquity UPLC C18 BEH (100 × 2.1 mm, 1.7 µm).

Guard column (Van Guard 5 × 2.1 mm, 1.7 µm, Waters).

Empower2 Pro software (instrument control and the data acquisition).

CHROMATOGRAPHIC CONDITIONS

Binary gradient elution program using water (A) and ACN (B).

Time(min) Water (A) % ACN (B)%

0-3 50-40 50-60

3-6 40-30 60-70

6-7 30 70

7-12 30-10 70-90

12-13 - 100

13-18 0-50 100-50

RESULT &

DISCUSSION

IMPORTANT POINTS

1. Optimization of the chromatographic conditions.2. Optimization of the SALLE conditions. Study of honey concentration. Study of the type and volume of the

extraction solvent. Study of the mass-separating agent and its

amount. Study of the extraction time and centrifugation

speed.3. Method validation. Selectivity, linearity, LOD & LOQ. Accuracy and precision. Uncertainty.4. Analysis of honey samples.

CHROMATOGRAPHIC CONDITIONS

i. COLUMNs : Acquity BEH UPLC C18 column. Phenyl columns ii. MOBILE PHASE:Water and acetonitrile.iii. TEMPERATURE: 25 °C – 35 °C Optimum iv. RESOLUTION: Resolution > 1.5 (Majority of the analytes) R (B[b]F–B[k]F ) = 1.2 R (Chry–B[a]A ) = 0

Where, B[b]F–B[k]F = Benzo(b)fluoranthene + Benzo(k)fluoranthene Chry–B[a]A = Chrysene + Benzo(a)anthracene

COLUMN: Phenyl stationary column. An improved resolution between B[b]F & B[k]F was obtained; however the lately eluted PAHs (namely Db[a,h]A, B[g,h,i]P, I[1,2,3-cd]P) were partially overlapped.

Finally, the gradient elution program was set on the C18 column was selected since the majority of the compounds are well resolved.

The compounds Chry–B[a]A were unable to separate and were eluted at a single peak. For this reason these compounds were measured as total “Chry + B[a]A”.

1. SALLE CONDITIONSSALTING OUT PHENOMENON:i. More polar solvent favorably surrounds the electrolyte

(salt) due to the Coulombic forces. ii. One of the solvents preferentially solvates the electrolyte

(ionic or not ionic) making it unavailable to dissolve the other solvent].

It is possible the above phenomena can act synergistically.

FACTORS ON WHICH SALLE: iii. The solvent type and volume iv. Salt type and amountv. Sample concentration which are needed to be examined

and optimized in order to establish the optimum working conditions.

POINTS TO PONDER Honey is sugar rich it contains monosaccharides, oligosaccharides, in total of ca 77% its matrix may interfere the performance of SALLE due to the sugaring-out effect .So extraction procedure were optimize in SALLE parameters using a PAH-free pooled honey sample (n = 5) spiked at concentration level of 10 g L−1 of the analytes. The extraction recovery(ER, %) and the enrichment factor (EF) were calculated.PAHs tend to absorb on the wall of the containers their potential retention on the syringe filter during the filtration process of samples using PAH standard solutions in ace-tonitrile at level of 10 g L−1 were examined.The peak areas obtained were no statistically different (at 99%

confidence interval) using the t- student test.

i. HONEY CONCENTRATION Sample concentration = 100–500 g L−1 . Solvent = 2500 L of ACN as hydrophilic solvent Salt = 1g NaCl as mass-separating agent The maximum = 200 and 250 g L−1 (RSD < 7%, n = 6) The sample concentration of 250 g L−1 was finally

selected. It should be noted that during SALLE extractions a thin

and white middle layer (containing polysaccharides, proteins) was observed at the interface between the aqueous and ACN layers that did not influence the collection of the PAH-rich phase

Lower preconcentration factors were obtained at elevated sample concentration (>300 g L−1 ).

This maybe attributed to the higher sample solution viscosity preventing the mass transference of the analyte to the organic layer.

At the level of 500 g L−1 the resulted solution was highly viscous hindering the penetration of the added ACN in the sample while phase separation was achieved without the addition of salt .Additionally, the ACN layer was colored yellowish indicating that some constituents were extracted from the honey matrix that could potentially interfere the chromatographic analysis.

• In SALLE the solvent has to meet some general criteria:

i. High extraction efficiency.ii. Good chromatographic behaviour .iii. To form a consistent clear layer after phase

separation preferably at minimum salt amount. TYPE OF SOLVENT: In this experiment, Methanol (5.1), ethanol

(5.2), ace- tonitrile (5.8), acetone (5.1) and isopropanol (3.9) were examined.

ii. TYPE & VOLUME OF SOLVENT

• VOLUME OF ACN:• The obtained results showed that higher

extraction recoveries (> 60%) were achieved by using ACN than those of the other solvents.

• A series of experiments was performed altering the added ACN volume in the range of 1500–2500 L since lower volumes resulted to difficulty on the retrieval of the ACN volume due to the formation of polysaccharides’ layer.

• When selecting an electrolyte for the salting-out processes.

• The salt should be negligibly soluble in the water-miscible organic solvent.

• Freely soluble in water in order to have the maximum interaction with water molecules and

• The salting- out capacity follows the Hofmeister series

iii. MASS SEPARATING AGENT

The effect of various salts including NaCl, MgSO4 , Na2 SO4 , (NH4 )2 SO4 and ZnSO4 on the performance of the proposed method were studied.

According to literature the salt’s anion is predomi- nantly responsible for the efficient phase separation and it follows the Hofmeister series (SO4 2− > Cl−).

NaCl was finally selected for subsequent studies due to its strong salting-out ability and high solubility in aqueous samples.

AMOUNT OF SALT

The effect of the added NaCl amount was investigated in a range of 0.5–2.0 g in the 5 mL of honey sample solution.

At Higher amounts NaCl would be oversaturated in the solution so have to increase ACN volumes which causes dilution.

At low amount of salt (ca. 0.5 g) poor phase separation was observed while the retrieval of ACN layer was difficult leading to high uncertainties. 1 g of NaCl was finally chosen as it provides

adequate sensitivity and reproducibility.

Two ways were used .i. Ultrasonic treatment.ii. Manual shaking

i) ULTRASONIC: Poor precision (>16% RSD) due to the

incomplete salt dissolution. ii) MANUAL SHAKING :• The manual shaking-based extraction was

adopted.• Extraction time = 1 to 10 min.

EXTRACTION TIME

• For all analytes the EFs were sharply increased from 1 to 5 min and then remain almost unaffected. • This designates that the diffusion of PAHs from honey sample to the ACN medium is relative fast

despite its high viscosity.

Thus the extraction time of 5 min was selected.

The effect of centrifugation speed on the extractability of the PAHs was examined in the range of 3500–4500 rpm at con- stant centrifugation time of 5 min.

The averages of peak areas were slightly improved up to 4000 rpm and being unaffected at higher rates. Therefore, a centrifuge speed of 4000 rpm was

chosen and adopted.

CENTRIFUGATION SPEED

METHOD VALIDATION

i. Specificity.ii. Sensitivity.iii. Linearity.iv. Limit of detection (LOD).v. Limit Of Quantification (LOD). vi. Precision (repeatability and intermediate

precision).vii. Trueness (in terms of recovery) and uncertainty.

SELECTIVITY

20 blank honey samples from different floral regions were examined to check the presence of potential interferences.

No interfering peaks present in the elution region of the analytes of interest were observed considering the sufficient selectivity for the trace analysis of PAHs.

LINEARITY1. Calibration Curves Method Sample studied in the range of 0.1 to 20 g L−1

(corresponding to 0.4–80 ng g−1 of the target analytes in the honey samples) by plotting the peak area of the each analyte against to the concentration. For each calibration level five independent sample extractions were carried out.

2. Homoscedasticity test (expressed as F-value): It revealed that there was a significant difference

between the variances obtained at the lowest and at highest calibration level (Fexp > F4,4,0.99 = 15.98)) at the confidence level of 99% for all analytes . For all compounds, the coefficient of

determination was greater than 0.99 demonstrating

satisfactory linearity in the concentration range studied.

LOD & LOQ

LINEAR REGRESSION METHOD:The param eters of the linear regression equations: i. Slope (a).ii. Intercept (b).iii. Standard deviations of the slope (Sa ).iv. Standard deviations of the intercept (sb ).v. Correlation coefficient (r)vi. LOD .vii. LOQ.

Matrix-matched regression equations = 3 sb /a & 10 sb /a.The LOD achieved for the target PAHs were in the range of 0.02–0.04 g L−1 (equivalent to 0.08–

0.16 ng g−1 in the honey sample) which are lower than the maximum residue limits for B[a]P, B[a]A, B[b]F and Chry (0.3 ng g−1 each) as established by European Commission EC No 836/2011 [32].

Matrix-matched regression

ACCURACY

Closeness to the true value

a. Sample = Honey.b. Experiment = A pooled blank honey sample

fortified with the target analytes.c. Five individual extractions (m = 5) were

performed per each concentration level (0.4, 2, 4 and 40 ng g−1 ) (p = 4) at five consequent working days (q = 5).

Percent Recovery :

R% = (Cspiked /CSTD ) × 100Where, Cspiked =Experimentally found concentration of the

analytes.

CSTD= Theoretical spiking concentration level . The Cspiked was calculated from the matrix-matched calibration curves using the respective chromatographic peak areas.

ANOVA ( One way variance method)

It was employed to deter- mine the mean recovery at each spiking concentration level (5 × 5 spiked samples), the repeatability and between-day reproducibility of the method. As it can be seen on Table 3 the mean recovery at the all studied levels was varied from 74 to 113% which fully complied with the recommended ranges for trueness based on Commission Decision 2002/657/EC.

The method repeatability and intermediate precision were also investigated via ANOVA calculating the F-values. In this test the between days variance over the within-day one was assessed. In all cases the experimental Fexp values were lower than theoretical Ftheor (F4,12 , 0.05) = 3.26). As it shown in Table 3 the mean repeatability (expressed as RSDr %) and between-days reproducibility (expressed as RSDR ) were satisfactory being in the range of 6–17% and met the criteria of Commission Decision 2002/657/EC [21].

HORWITZ RATIO & HORRAT VALUE (H)

A relationship between the variability of chemical measurements and the concentration of the analyte.This relationship, called the Horwitz curve applies only to between-laboratory variability of measurements.

H = RSDR /RSDH Where, RSDR = Experimental relative standard deviation of

the within-laboratory reproducibility. RSDH = “target” relative standard deviation. It should be ranged between 0.2 and 1. For concentrations lower than 120 g Kg−1 , the “target”standard deviation

(sH ) and therefore the “target” relative standard deviation RSDH is given as sH = 0.22 C

UNCERTAINITYUNCERTANITY:

Where, u(P) = Method precision uncertainity. U(Rm)= Recovery uncertainity.

TOTAL UNCERTAINITY:Where, u(P)/P & u Rm /Rm are the contributions of the within- laboratory

reproducibility,

t -TEST VALUE:

EXPANDED UNCERTANITY:

No detectable levels of the studied compounds were found this could be attributed to the low environmental pollution in the geographical region or the accurate smoking process.

ANALYSIS OF SAMPLE

Benzo(a)pyrene (B[a]P ) :

High B[a]P level has been found in other beehive products (e.g. propolis) as reported.

The applicability and accuracy was demonstrated by spiking the samples at different concentration levels in the range of 0.1–1.0 g L−1 (0.4–4 ng g−1 ).

The average recoveries for all analytes ranged from 51 to 118% .

The relative standard deviation was lower than 19%.

B[a]P, B[a]A, B[b]F and Chry

The recoveries were between 56 and 116% in all studied fortification levels which is in compliance with the criteria established by Commission Regulation (EC) No836/2011 .

A representative chromatogram of a spiked honey sample at concentration level of 0.1 g L−1 (0.4 ng g−1 ) of all the compounds is illustrated in Fig. 5.

The proposed SALLE method is feasible for quantitative analysis of the selected PAHs in real samples, and could be used in routine analysis.