PII: S0025-326X(98)00170-2

Marine Pollution Bulletin Vol. 38, No. 3, pp. 193201, 1999 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0025-326X/99 $ see front matter

Revisiting the Development of the Bligh and Dyer Total Lipid Determination Method
FOPPE SMEDES* and TORSTEN K. ASKLAND Ministry of Transport, Public Works and Water Management, National Institute for Coastal and Marine Management/ RIKZ, P.O. Box 207, 9750 AE Haren, The Netherlands

The experiments leading to the development of the most well-known method for total lipid determination in marine biological tissues (Bligh, E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911917) were repeated in order to discover the secrets of its success. Along with measuring the phase volumes of the water/methanol/ chloroform mixtures investigated by Bligh and Dyer, the phase compositions were determined by gas chromatograph (GC). An examination showed that, although Bligh and Dyer applied largely dierent solvent ratios, the composition of both phases varied only within a limited range resulting in an incomplete investigation of the effects of changing this factor. Using Bligh and Dyers solvent mixtures the recovered fraction of organic phase was found to be the key factor determining the extracted lipid yield. On its turn the recovered fraction was positively correlated to the size of the organic phase and its methanol content. Additional experiments applying new extraction points with higher methanol contents revealed an increase of extracted lipid. This increasing yield was mainly due to a better extraction of the phospholipids as could be deducted from lipid patterns recorded by normal phase high performance liquid chromatography (HPLC) using an evaporative mass detector. 1999 Elsevier Science Ltd. All rights reserved. For studies of pollution levels in the marine environment the lipid content of biological material is a crucial parameter to interpret data on organic contaminants (Schneider, 1982; Delbeke et al., 1995). Lipid is a natural mixture of triglycerides, diglycerides, monoglycerides, cholesterols, representing the more apolar or neutral lipids and free fatty acids, and phospholipids, sphingolipids, etc. representing the polar lipids (Lovern, 1957). Solvents to extract lipids must demonstrate a high solubility for all lipid compounds and must be suciently polar to remove the lipids from their association with
*Author to whom correspondence should be addressed. Present address: LEO Pharmaceutical Products, Industriparken 55, DK-2750, Ballerup, Denmark.

cell membranes and lipoproteins. Chloroform/methanol mixtures apply well as was recognised by Folch et al. (1957). This approach was adapted by Bligh and Dyer (1959) resulting in a method which has become the standard method for total lipid determination for over 30 years. Chemists claiming they use the Bligh and Dyer method for lipid extraction usually apply a modication of this method (de Boer, 1988; Booij and van der Berg, 1994; Gardner et al., 1985). The evaluation of an intercalibration exercise revealed that, to some extend, dierences in the outcome could be related to deviation from the original method (Roose and Smedes, 1996). When laboratories agree to apply exactly the same procedure results became highly comparable (Randall et al., 1991). In a previous paper (Smedes and Thomasen, 1996) the original work of Bligh and Dyer was evaluated from a theoretical viewpoint. The variable lipid contents found by Bligh and Dyer applying eleven dierent solvent mixtures could not be explained by dierences in the ability to dissolve lipids nor adsorption to the tissue residue. Further reasoning revealed that the measured lipid content was mainly determined by the fraction of organic phase that could be recovered. The methanol contents in the organic phase varied only little and the optimum methanol content cannot be derived from the Bligh and Dyer experiments. However an indication was found that methanol had a positive eect on the extraction yield, either because less organic phase was sticking to the tissue or better extraction kinetics, due to higher solubility in the mono-phasic situation and in the aqueous phase. The order solvents were added also seemed important for the kinetics of the extraction. First the association with cell constituents is destroyed where after lipids are dissolved in a mono-phasic system. Then transfer to an organic phase is performed in a bi-phasic system created by the addition of more chloroform and water. Although a positive eect on extraction kinetics is plausible no experiments are known comparing one-step and two-step extractions using the same solvent ratios. 193

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The present paper reports about a complete repetition of the experiments as performed by Bligh and Dyer. In addition one-step extractions are compared with twostep extraction and the inuence of solvent compositions on the extraction yield is further investigated. The patterns of extracted lipids were investigated by High Performance Liquid Chromatography (HPLC) with an evaporative mass detector (EMD).

Materials and Methods

Chemicals Chloroform (Merck, Darmstadt, Germany) and methanol (Baker Chemicals, Deventer, The Netherlands) were both of Pro Analyse quality. Water was delivered by a Milli-Q system (Millipore). The lipid standards (Sigma, St. Louis, MO, USA) used were: Cholesteryl palmitoleate (CHOLE), acyl-sn-glycero-3phosphocholine (LPC), Trioliene (TG), Oleic acid (FFA), Cholesterol (CHOL), 1,2-diacyl-sn-glycero-3phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3phosphocholine (PC), Sphingomyelin (SM) and 1,2diacyl-sn-glycero-3-phosphoserine (PS) all of high purity (>99%). Other solvents used were hexane and tetrahydrofuran (Baker Chemicals, Deventer, The Netherlands), both HPLC quality.

by 3 min mixing. This mixing was repeated for 30 s both after adding the second portion of chloroform and after addition of water. After centrifugation the organic phase was isolated using a Pasteur pipette and the weight was recorded. This extract was split in two parts on weight basis. One part was transferred to an aluminium cup, evaporated to dryness and the weight of the residue was determined after 30 min at 105C. The other part was ltered by means of 13 mm 0.5 lm PTFE lter (Millex, LCR13, Millipore), concentrated to obtain a lipid concentration of 50 mg/ml and used to record a lipid pattern by HPLC-EMD. Applying 4 min of mixing the Bligh and Dyer extractions AE were repeated without a stepwise addition of solvents. This procedure was also applied to an extra set of extractions, here called KN wherein the methanol amount was varied complementary with the water keeping the total volume constant. Gas chromatographic analysis To analyse the solvent compositions a Varian 90P gas chromatograph (GC) was used equipped with a thermal conductivity detector. Helium was applied as a carrier gas at a ow of 140 ml/min and the compounds were injected at 250C on a Porapak-Q column (stainless steel, lengthdiameter was 20002.1 mm) at an oventemperature of 140C. Chromatographic data were processed by computer utilising Turbochrom (Perkin Elmer) software. Calibration was performed by injecting standard solutions and quantication was done on peak area. HPLC analysis Lipids were separated with a normal phase column connected to a Hewlett Packard 1050 high performance liquid chromatographic system consisting of a solvent degasser, a quaternary pump (ow rate 0.4 ml/min) and an automatic injector. The detector was an evaporative mass detector (PL-EMD 940, Polymer laboratories, UK) used at a temperature of 55C and an airow of 10 l/min. In the detector the eluent is sprayed with air in a heated tube where it evaporates. Non evaporating compounds in the eluent remain in the air stream as small droplets that are detected by light scattering. Standards and extracts were injected on a Chromsphere CN column (1503 mm, 5 lm, Chrompack , The Netherlands) either in chloroform or toluene. The gradient used started with 2% tetrahydrofuran in hexane and after 0.5 min this was linearly programmed to be 86% at 6.5 min. In the next minute the composition is programmed to 50/50 tetrahydrofuran/methanol and subsequently to 100% methanol from 7.5 to 8.5 min. Elution with methanol is maintained till 15 min and then the gradient is programmed back to 100% tetrahydrofuran and nally back to the initial composition. To obtain equal column activity and subsequently constant retention times a xed equilibrium time (30 min) was required before each injection. An automatic injector is

Sample Preparation and Extraction

About 1 kg cod iced llet from fresh sh was purchased from a local supplier and quickly homogenised in a 1 liter glass jar placed on ice using an Utra Turrax, T45 (Janke & Kunkel kg, Staufen, Germany). From this homogenate portions of 5 and 10 g were weighed in 50 glass jars of 100 ml with a polypropylene lid. During homogenisation the temperature did not exceed 10C. Samples were stored in a freezer at20C until extraction. For extraction an Ultra Turrax mixer T25 (IKA Labortechnik) with a 18 mm shaft was used and phase separation was achieved by centrifugation for 10 min at 2000 rpm at 20C in a thermostated centrifuge (Sigma 3K12, Germany). All extractions were performed in the same way as Bligh and Dyer in 1959, except that sample and solvent amounts were reduced by about a factor 10. Solvent additions were done on weight basis, while also total weights were recorded throughout the whole procedure to detect possible evaporation or spilling. During extraction the weight decreased approximately 0.6 g due to evaporation and in two cases some mixture was lost by splashing. The loss represented 12% for the higher volumes and up to 3% for the smaller volumes. Upscaled, but otherwise identical, blank extraction mixtures were also put together and weight and volumes of the formed phases were determined as well as the solvent composition. The extraction procedure is briey as follows: methanol and chloroform were added to the sample followed 194

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prerequisite to accomplish this. The rst injection should be ignored since it will always have a deviating equilibration history. Chromatographic data were processed as for GC analysis and quantied by peak area.

yield from the mass balance according to Smedes and Thomasen (1996) is given by the mass balance T: m gOrg wOrg gAq wAq gTis sTis Y 1 where m is the total amount of lipid, COrg , CAq and CTis are the lipid contents respectively in the organic-, waterand tissue phase, and MOrg , MAq and ITis are the amounts of the subsequent phases. To relate the lipid content in the organic phase, COrg , to the amount of organic phase, MOrg , eqn (1) is rewritten as follows: 1 sTis wOrg gAq wAq gTis sTis X m sTis gOrg gOrg gOrg 2

Results and Discussion

In Table 1, part I, the solvent weights used (in accordance with the experiments of Bligh and Dyer) are listed. Solvents were added on weight basis and calculated back to the equivalent for 10 g intake although in some cases only 5 g sample was processed. Honeycut et al. (1995) evidenced that scaling down to 5 g did not inuence the extractable weight. The added solvent weights for the extra experiments are given in part II of Table 1. For all extractions the recovered organic phase, the lipid content determined from that organic phase and the organic phase volumes measured from blank mixtures are listed. The phase compositions determined from the blank mixtures are given in Table 2 and were in agreement with earlier work (Smedes and Thomasen, 1996). A check of a few mixtures in the presence of sample showed that compositions were not inuenced. The obtained data are evaluated below in relation to the dierent variables. Multiple-step extractions In the extractions A through E solvent compositions are extremely close while the organic phase volume is increasing over a factor 10. At the same time the aqueous phase varies only a factor 2 what allows one to study the inuence of the organic phase volume on the lipid

In this equation the last two terms are nearly constant. The amount of aqueous phase varies only little in the second last term and the ratio between the concentration in aqueous and organic phase equals the partition coecient, which is a constant provided the solvent composition is xed. For the last term a similar reasoning is valid. In the formula ITis /m equals the reciprocate value of the lipid content in the tissue (CL ). Regression analyses with l/COrg as y-variable and the amount of organic phase per gram tissue, MOrg /ITis , as x-variable will return the reciprocate value of CL as the slope. When an intercept is detected the extraction is not complete and aqueous phase or tissue still contains lipid. In Fig. 1 the above mentioned variables are plotted and regression analyses of the repeated Bligh and Dyer experiments revealed a non-signicant (P>0.3) negative intercept. The regression line drawn in Fig. 1 is therefore forced through zero. The absence of an intercept proves that, neither the solubility of lipids in the aqueous phase nor sorption onto the tissue is of any signicance. Be-

TABLE 1 Weights of solvents applied for multiple step lipid extractions as performed by Bligh and Dyer (part I) and additional experiments (part II). Furthermore the recovered organic phase and the lipid content measured in it are given. For each extraction the weight of the organic phase measured from a blank mixture is given. I Bligh and Dyer extraction mixtures Weights of solvents in rst step (g) Weight in second step Weight in third step Recovered organic phase (g) Lipid content in organic phase (mg/g) Organic phase in blank mixture (g) Lipid content in tissue (mg/g) II Additional single step extractions Weights of solvents Recovered organic phase (g) Lipid content in organic phase (mg/g) Organic phase in blank mixture (g) Lipig content in tissue (mg/g)
a b c

A Methanol Chloroform Waterb Chloroform Water 11,8 3,5 8,0 9,6 17,0 2,2 8,52 8,3 7,03 A Methanol Chloroform Water 11,8 9,8 17,1 1,8 7,57 8,3 6,29

B 16,4 8,2 8,0 23,9 24,1 14,3 3,27 22,6 7,40 B 16,4 24,0 23,9 14,4 2,65 22,6 5,97

C 18,6 14,3 8,0 39,4 27,0 30,8 1,93 38,4 7,42 C 18,6 39,3 27,0 29,5 1,74 38,3 6,65

D 21,9 24,5 8,0 67,6 31,5 56,2 1,07 67,2 7,18 D 22,0 67,5 31,5 57,7 0,95 67,2 6,37

E 28,9 44,8 8,0 88,7 41,5 71,4 0,80 88,5 7,05 E 29,0 88,9 41,6 76,2 0,75 88,7 6,63

F 9,8 14,7 8,0 11,0 5,55 13,8 7,64 Kc 0,0 30,4 39,0 30,4

Ga 14,2 7,6 16,4 1,3 17,2 7,7 13,2 L 8,2 30,6 29,0 17,3 1,42 30,5 4,32

H 7,1 8,2 8,0 3,4 10,3 7,4 7,57 M 12,2 30,3 23,6 20,7 2,00 30,4 6,09

I 6,5 14,5 8,0 9,8 5,11 14,2 7,26 P 16,4 30,4 18,5 25,3 2,35 29,6 6,96

J 6,2 26,5 8,0 21,7 2,55 26,7 6,81 N 17,9 30,2 16,3 24,6 2,56 31,5 8,06

P 16,4 15,2 8,0 30,4 18,4 23,5 2,41 29,6 7,14 O 20,3 30,1 13,2 22,3 2,51 33,2 8,36

Strong emulsion, organic phase dicult to recover. First 8 g of water origins from the sample. No recovery of organic phase possible.

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Marine Pollution Bulletin TABLE 2 Actual solvent compositions (in % w/w) of both phases in blank extractions. Aqueous phase Water A B C D E F G H I J P K L M N O 58.2 58.9 59.4 59.4 59.4 45.4 52.2 52.4 56.0 58.6 53.3 99.7 77.3 65.8 48.5 38.5 Methanol 38.6 37.7 37.8 37.3 37.3 45.2 43.1 42.4 39.7 37.6 41.6 0.0 21.4 32.0 43.8 45.7 Chloroform 3.2 3.4 2.8 3.3 3.3 9.4 4.8 5.2 4.3 3.8 5.0 0.0 1.3 2.2 7.8 15.8 Water 0.4 0.4 0.4 0.3 0.3 2.4 1.0 0.9 0.5 0.3 0.8 0.0 0.1 0.2 1.5 4.7 Organic phase Methanol 3.8 3.5 3.3 3.1 3.1 12.1 7.1 6.3 4.5 3.4 6.0 0.0 0.7 2.0 9.6 17.9 Chloroform 95.5 96.0 97.1 96.9 96.9 85.6 93.2 93.1 95.5 96.1 93.0 99.0 98.6 97.6 88.8 77.1

cause of this outcome, the fact that the aqueous phase volume was not constant becomes irrelevant too. The extraction of lipids seems a simple solubility process and for all extractions, AE, the lipids were equally sucient extracted to the organic phase. In Fig. 1 the slope of the regression line implies a lipid content of 7.14 (0.06) mg/g wet weight [see eqn (2)]. The increasing lipid yields from A through E found by Bligh and Dyer are not related to dierent solvent compositions but entirely due to increasing recovery of organic phase. A larger organic phase results in a more complete recovery. Assuming that the lipid yield for the experiments AE found by Bligh and Dyer reects the recovered organic phase, it can be calculated that the yield of organic phase is 818% lower for the experiments described in this report. This can easily be explained by the dierence in procedure. Bligh and Dyer ltered the tissue and

squeezed it for maximal yield. Apparently squeezing is more eective to obtain maximal yield than centrifugation. This paper was focusing on the determination of Corg and because solvent evaporation cannot be controlled during ltration, centrifugation in closed jars was more appropriate to obtain phase separation. Single step extractions Concluding that the lipid yield is predominantly determined by the recovery of the organic phase, the importance of using a multiple step approach for the extraction becomes questionable. The lower lipid contents found by Bligh and Dyer for FJ cannot unambiguously be attributed to the fact that extraction was performed in a single step. Therefore these single step extractions were repeated and, in addition, also single step extractions were performed with the mixtures AE. Lipid contents calculated from COrg and the organic phase volume from blank mixtures were summarised in Fig. 2. The open squares represent the lipid contents for a single extraction and the solid squares the multiple step extractions. A horizontal dotted line represents the lipid content calculated above by linear regression (Fig. 1). The vertical bars indicate the water respectively the methanol contents in the organic phase. The extractions AE clearly show a higher lipid yield for the multiple step approach, which means that a stepwise addition of solvents promotes the extraction kinetics. F, G, H and I, however, demonstrate that a single step extraction can result in a high yield too. The G extraction is an artefact here. Due to a dicult phase separation, hardly any organic phase could be recovered (<1 ml) what made weighing very inaccurate. For the P extraction a single step extraction performs nearly equal to multiple step. Based on observed precipitation Bligh and Dyer concluded that co-extraction of non-lipids took place for the F extraction. Although precipitation occurred in-

Fig. 1 Relation between the reciprocal lipid content in the organic phase (y-axis) and weight of organic phase applied per gram sample. No signicant intercept was found and therefore the line is forced through zero. The slope equals the reciprocal lipid content i.e. 7.14 (0.06) mg/g.

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Fig. 2 Lipid contents (left y-axis) in cod esh using dierent solvent mixtures as indicated on the basis. The lled squares (n) represent the results using multiple extraction. Values obtained by single step extraction are shown by an open square (h). A dotted line is drawn at the lipid content found by regression (Fig. 1). The methanol and water contents in the organic phase are indicated by respectively open and closed bars (right y-axis).

deed, not co-extraction but azeotropic distillation was responsible for this eect. Because chloroform forms an azeotrope (Weast, 1979) with methanol of 13% (v/v), evaporation of the organic phase of F (20% methanol) ends in water/methanol what causes lipids to precipitate. Repeating the evaporation after addition of extra chloroform results in a clear solution. Since for F, H and I the lipid contents are equal or higher than P the multistep approach does not seem to be obligatory for a quantitative extraction in all cases. Methanol content As mentioned before the methanol contents did not really vary in the Bligh and Dyer experiments. However the results in Fig. 2 do indicate a positive inuence of the methanol on the extraction yield although phase volumes varied at the same time. Therefore the extractions KO were performed which, together with P, cover the entire possible range of 020% (w/w) methanol in chloroform. Addition of more methanol results in a mono-phasic system. In the extractions the amount of chloroform used was kept constant. Without addition of methanol (situation K) a very strong emulsion was formed, which could not be broken even by prolonged centrifugation. As a result no organic phase could be recovered. In Fig. 3 the measured lipid yields (solid squares) are plotted against the methanol content and show a linear relation with the log methanol content. It appears that

composition P, selected by Bligh and Dyer, does not give the optimum yield, although the possible increase in yield at higher methanol contents is not very large. Considering the importance of the methanol content all the other obtained lipid yields are also shown in Fig. 3. All single-step extractions, indicated by a squareframed letter, are grouped around this line and seem to follow the same relation as LO. The multiple-step extractions, indicated with a circled letter, show a higher yield than expected from their methanol content. Apparently the much higher methanol content they were subjected to in the rst step, i.e. around 50% which is higher than O, dissolves lipids which are not (completely) re-adsorbed when in the following steps the methanol content is decreased again by addition of more chloroform and water. This elevated extraction yield at higher methanol contents explains the better extraction capability of multiple-step procedures and conrms the view of Bligh and Dyer on this. Lipid patterns with HPLC Besides determining the weights of the extracted lipid material, all extracts were analysed with HPLC to determine the lipid composition. For the separation of lipids, Christie (1985) used normal silica and a gradient from isooctane/isopropanol, via isopropanol/chloroform, ending in isopropanol/water. The necessity to apply water to elute the phospholipids is a severe disadvantage as it needs extensive washing to activate the column for the next analysis. A CN bounded column 197

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Fig. 3 Presentation of the lipid contents in relation to the methanol content (log scale). The lled squares are the one step extractions performed to see the inuence of the methanol content. All other extractions, indicated by a letter, are shown too. Squares are the one-step and circles are the two-step extractions.

allows elution of phospholipids with only methanol. Using hexane, tetrahydrofuran and methanol the lipids could be separated in the dierent groups. It should be noted that also with this type of gradient the separation is strongly inuenced by the activity of the column and the equilibration period before each injection should be remained constant. The use of an automated injection and gradient system is therefore inevitable. In Fig. 4 a typical chromatogram is presented. The upper one is a 10 times expansion of the lower one in order to show the details. The CHOLE and LPC elute at the same time and are only partly separated from the largest TG peak. Two medium large peaks were attributed to FFAs. Although most lipid peaks are not pure compounds (dierent alkyl groups) this is not true for the cholesterol, which is a pure compound. Diglycerides show two peaks; the 1,2 acylated and the 1,3 acylated glycerol. The same distinction occurs for monoglycerides; acylation at the 1 or at the 2 position of the glycerol. All phospholipids elute at the end of the chromatogram and are separated in a peak representing PE, another representing the PC and SM, which elute together, and a small one representing the PS. From the chromatogram one can see that the phospholipids represent the major fraction in cod esh followed by cholesterol, the triglycerides and free fatty acids. In Table 3 198

the measured concentration of the dierent lipid groups are given for all extracts. The contents of minor constituents are not very accurate. Although the chromatogram shows nice peaks without any baseline noise one should consider that the signal is the result of light scattering of small lipid droplets. Response diminishes rapidly when droplets get small and their diameter range approaches the wavelength. Secondly, not only the solvent evaporates in the detector but, depending on the properties, also the compounds of interest can evaporate. This evaporation is independent of the amount present and is only inuenced by the detector temperature and air ow. The result of both eects is that the amount of compound has to exceed a certain threshold and the obtained signal is only a measure of the amount above it. Furthermore the signal of an EMD is not linear with the concentration. These shortcomings can be accounted for by using a second order calibration polynome, but nevertheless a relatively high error can occur especially for the lower contents. Total lipids by HPLC-EMD can be calculated by adding up. A comparison of the yield found by evaporation to dryness with the sum of lipids determined by HPLC reveals that the latter are slightly but consistently higher, 13% (2.6). This is probably an eect of the calibration although it is also possible that

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Fig. 4 Typical HPLC chromatogram. The upper one is a 10 times expansion of the lower one. For abbreviations see text.

solvents evaporate dierent from mixed lipids in sample than from the pure lipid compounds in standards. Incomplete evaporation will cause larger droplets and consequently more scattering. Al these uncertainties were, to a large extend, surpassed by adjusting the injected amount to about the same level for all the samples, so the underlying dierence would not hamper the comparison of the dierent extractions. Lipid composition Evaluating the lipid compositions in Table 3 learns that no spectacular dierences are present between the

extractions as applied by Bligh and Dyer. A deviating composition is only observed for the one step extractions with lower methanol contents (L and M). Especially extraction of the phospholipids is incomplete while the neutral lipids and cholesterol show much less dependency on the methanol content. This is visualised by a bar graph in Fig. 5 where the sum of glycerides and cholesterol for the dierent methanol contents are compared with the sum of the phospholipids (note the dierent scales). The presence of methanol especially favours the extraction yield of the latter group while the glyceride group is nearly equal for each methanol con-

TABLE 3 Individual lipid contents in the dierent extracts in mg/g extract. For abbreviations see text. The last two columns on the right allow comparison of the total content measured by HPLC and gravimetrically. CHOLE/LPC AS BS CS DS ES FS GS HS IS JS PS LS MS NS OS AM BM CM DM EM PM 0.06 0.05 0.06 0.05 0.06 0.11 0.07 0.06 0.05 0.06 0.05 0.06 0.06 0.07 0.07 0.06 0.07 0.07 0.06 TG's 0.34 0.32 0.37 0.30 0.39 0.40 0.74 0.45 0.41 0.37 0.37 0.32 0.33 0.45 0.48 0.39 0.40 0.41 0.39 0.43 0.37 FFA's 0.32 0.34 0.41 0.36 0.22 0.29 0.64 0.35 0.37 0.30 0.34 0.33 0.35 0.27 0.32 0.44 0.43 0.44 0.42 0.43 0.60 CHOL 0.38 0.36 0.40 0.35 0.43 0.44 0.77 0.48 0.43 0.44 0.40 0.33 0.42 0.48 0.47 0.41 0.41 0.39 0.37 0.38 0.39 DG's 0.07 0.06 0.07 0.07 0.08 0.08 0.14 0.08 0.07 0.07 0.06 0.06 0.07 0.06 0.07 0.06 0.07 0.06 0.06 0.06 0.06 MG's 0.04 0.04 0.04 0.04 0.04 0.04 0.07 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.05 PE 1.0 1.0 1.1 1.0 1.1 1.2 1.8 1.2 1.2 1.1 1.1 0.7 1.0 1.2 1.2 1.1 1.1 1.1 1.1 1.1 1.0 PC/SM 4.7 4.6 5.1 5.0 4.9 5.9 10.4 6.0 5.7 5.3 5.4 3.0 4.6 6.4 6.7 5.7 6.1 6.1 5.6 5.7 4.9 PS 0.07 0.10 0.12 0.15 0.17 0.19 0.14 0.13 0.12 0.14 0.07 0.16 0.09 0.08 0.12 0.19 Total 6.9 6.9 7.6 7.3 7.2 8.5 14.8 8.8 8.3 7.8 7.8 4.8 7.0 8.9 9.5 8.2 8.6 8.7 8.2 8.3 7.6 Gravi-metrical 6.3 6.0 6.7 6.4 6.6 7.6 13.2 7.6 7.3 6.8 7.0 4.3 6.1 8.1 8.4 7.0 7.4 7.4 7.2 7.0 7.1

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Fig. 5 Lipid compositions in relation to the methanol content. The narrow bars represent the glyceride related lipids together with cholesterol and refer to the left scale. Phospholipids are represented by the wide bars and refer to the right scale. Clearly the methanol content is of greater inuence on the yields of phospholipids than on that of glycerides and cholesterol.

tent. This nding is very important for the validation of existing lipid extraction methods and likewise for the development of new methods. Validation of an extraction method using tissue in which triglycerides are the major compounds is only of limited value and does not necessarily apply to all sample types.

Torsten K. Askland was able to work at the National Institute for Coastal and Marine Management/RIKZ through mediation of the International Association for Exchange of Students for Technical Experience.

Conclusions
The optimisation of the total lipid determination method done by Bligh and Dyer is limited to a variation of the ratio between the sample and organic phase volume. Small volumes of organic phase do extract the lipids just as ecient as the larger volumes but the fraction organic phase recovered is smaller what results in an apparent lower extraction yield. The proposed mixture P, however, is very close to the optimum yield but using a higher methanol content the yield can be increased by about 15%. Multiple step extraction positively inuences the yield but is overruled by higher methanol contents. As the extraction eciency of the individual lipids responds dierently to changes in the methanol content the determination of lipid composition is essential for the validation and development of lipid extraction methods. 200

Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology 37, 911917. Booij, K. and van der Berg, C. (1994) Comparison of techniques for the extraction of lipids and PCBs from benthic invertebrates. Bulletin of Environmental Contamination and Toxicology 53, 7176. Christie, W. W. (1985) Rapid separation and quantication of lipid classes by high performance liquid chromatography and mass (light-scattering) detection. Journal of Lipid Research 26, 507512. de Boer, J. (1988) Chlorobiphenyls in bound and non-bound lipids of shes; comparison of dierent extraction methods. Chemosphere 17, 18031810. Delbeke, K., Teklemariam, T., Cruz, de la, E. and Sorgeloos, P. (1995) Reducing the variability in pollution data : the use of lipid classes for normalization of pollution data in marine data. International Journal of Environmental Analytical Chemistry 55, 147162. Folch, J., Lees, M. and Stanley, G. H. S. (1957) A simple method for the isolation and purication of total lipids from animal tissues. Journal of Biological Chemistry 226, 497509. Gardner, W. S., Frez, W. A., Cichocki, E. A. and Parrish, C. C. (1985) Micromethod for lipids in aquatic invertebrates. Limnology and Oceanography 30, 10991105. Lovern, J. A. (1957) The Chemistry of Lipids of Biochemical Significance, 2nd edn, revised. Methuen, London. Randall, R. C., Lee, H., Ozretich, R. J., Lake, J. L. and Pruell, R. J. (1991) Evaluation of selected lipid methods for normalizing pollutant bioaccumulation. Environmental Toxicology and Chemistry 10, 14311436.

Volume 38/Number 3/March 1999 Roose, P. and Smedes, F. (1996) Evaluation of a lipid intercomparison by investigation of methodological dierences. Marine Pollution Bulletin 32 (8/9), 674680. Schneider, R. (1982) Polychlorinated biphenyls (PCBs) in cod tissues from the western baltic: signicance of equilibrium partitioning and lipid composition in the bioaccumulation of lipophilic pollutants in gill-breathing animals. Helgolander Meeresforschung 29, 6979. Smedes, F. and Thomasen, T. K. (1996) Evaluation of the Bligh & Dyer lipid determination method. Marine Pollution Bulletin 32 (8/ 9), 681688. Weast, R. C., ed. (1979) Handbook of Chemistry and Physics, 59th edn. CRC Press, Boca Raton.

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