Analyst, July 1995, Vol. 120 1937

Determination of Organotin Compounds in Marine Mussel Samples by Using High-performance Liquid Chromatography-Hydride Generation Inductively Coupled Plasma Atomic Emission Spectrometry

Paola Rivaro, Laura Zaratin, Roberto Frache and Ambrogio Mazzucotelli Istituto di Chimica Generale, Universita di Genova, Genova, Italy

HPLC-hydride generation-ICP-AES for the determination of organotin compounds is described. A detection limit of 7 ng for tin was obtained and the organotin species were completely separated within 7 min. The system has been used for the determination of organometallic compounds in marine mussel samples. Preliminary results obtained in laboratory experiments are presented. Keywords: Organotin compound; marine mussel; high-performance liquid chromatography; hydride generation; inductively coupled plasma atomic emission spectrometry

Introduction Organotin compounds are used in a number of commercial applications including biocide additives which are used in antifouling ship-paint formulations. About 69% of shipping vessels are still being painted with paints containing tributyli- tin (TBT) in spite of efforts to reduce their use.' Attention has been given to TBT, although other organotin compounds such as triphenyltin (TPT) have been employed as a co-toxicant with TBT in some long-performance antifouling paints.2

Tributyltin, released from the paint, has been found to affect many non-target organisms, particularly molluscs3~4 and gastropods.5.6 Concentrations as low as 2-3 ng 1-1 in the water can produce chronic toxic effects on oysters (shell deforma- tion), mussels (growth inhibition of larval and veliger stages) and marine gastropods (sterilization of females). In a marine environment, dibutyltin (DBT) and monobutyltin (MBT) are also present as a result of TBT degradation, but they are less toxic to aquatic organisms than TBT. The analysis of organotins in mussels, used as indicator organisms in monitor- ing programmes assessing the quality of marine environments, can provide a time-integrated estimation of these compounds in water.' Therefore, a detailed investigation to identify and analyse organotins in marine organisms is desirable.

Different methods have been proposed to separate and quantitate organotin compounds. The most commonly used separation technique is GC followed by detection with flame spectrophotometry,&lO electron capture (EC),11,12 and MS. 13-14 Gas chromatography methods have high separation power and can be connected to very sensitive detectors. Unfortunately, they require the conversion of ionic organotin species to volatile derivatives by using appropriate Grignard reagents. 15.16 The derivatization and clean-up procedures become problematic and longer times are needed for analyses. However, HPLC coupled with element-sensitive detection systems can be used as a separation technique (as conventional

HPLC detectors give a poor response to organotin com- pounds).

A number of papers describe the use of FAAS,17.18 ETAAS,19 and ICP-AES20 as detectors for HPLC. However, FAAS gives poor detection and ETAAS cannot provide a continuous recording of the HPLC eluate. Although ICP-MS is a very sensitive technique and can detect chromatographic eluate, it is still, unfortunately, difficult to control for naturally occurring samples.

ICP-AES, though able to record HPLC separation on-line, presents, as a limiting factor, the torch instability when organic solvents are used. The effect of the organic solvents on the plasma is generally to significantly reduce its excitation properties. By this means, the analytical signal may be reduced or the plasma may be extinguished.21

Moreover, the possibility for tin to form volatile hydrides allows an increase in sensitivity when spectrochemical tech- niques are used. In this work, we inserted a hydride generator (gas-liquid separator) between the HPLC column and the ICP-AES detector, to overcome the above-mentioned prob- lem and to increase the instrumental sensitivity.

Experiment a1

Reagents

The methanol used was of HPLC grade. Hydrochloric acid, ammonium acetate, sodium tetrahydroborate, tropolone (2- hydroxy-2,4,6-cycloheptatrienone) and dichloromethane were of analytical-reagent grade and were obtained from Sigma (St. Louis, MO, USA).

The water was de-ionized water from a Milli-Q system (Millipore, Watford, Hertfordshire, UK).

Standard solutions of MBT, DBT, TBT, and TPT were purchased from Aldrich (Milwaukee, WI, USA) (95-98% nominal purity). Working solutions were prepared daily, by diluting the stock solutions with methanol.

Apparatus

HPLC The HPLC instrument used in the following experiments was a Varian (Palo Alto, CA, USA) LC system 5000 equipped with a 200 p1 Rheodyne (Cotati, CA, USA) injector. Separations were performed on a Partisil (Whatman, Englewood Cliffs, NJ, USA) SCX 10 analytical column (10 pm particle size, 25 cm x 4.6 mm id). The flow rate was 1 ml min-* and no gradient elution devices were used. The mobile phase used at the beginning of the optimization of the HPLC separation was

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1938 Analyst, July 1995, Vol. 120

0.1 mol 1-1 ammonium acetate in 80% methanol-water. The pH was 7.4. The mobile phase was modified by adding 0.1% m/v tropolone.

ICP-AES ICP-AES was carried out using a Jobin Yvon 24 spectrometer (Jobin Yvon, Longjumeau, Paris, France) as the detector. The ICP-AES parameters were: power, 1.1 kW and an argon flow rate for intermediate, 16; coolant, 0.7; and nebulizer, 0.3 1 min-1. The HPLC-ICP determination was carried out detecting the tin signal every 300 ms with a scanning step of 0 nm, i .e . , the ICP detector was fixed at the analytical tin wavelength, Sn" 189.989 nm. The plasma conditions were established such that stable plasma operation was possible. A Jobin Yvon hydride generator was used as a gas-liquid separator.

The conditions for hydride generation were optimized. The effects of different concentrations of HCl and NaBH4 on the tin hydride formation were evaluated. The best sensitivity was obtained with 0.3 rnol 1-1 HCl and 0.25 mol 1-1 NaBH4 in 1 mol 1-1 NaOH with a 0.7 ml min-1 flow rate for both reagents.

Analytical Procedure

The eluent coming out from the HPLC column was mixed in a PTFE T-piece with hydrochloric acid, supplied by a peristaltic pump, with a 0.7 ml min-1 flow rate.

The organotin compounds were converted into their hydrides by addition of NaBK, supplied by a peristaltic pump with a 0.7 ml min-1 flow rate, into another PTFE T-piece. Evolved hydrides were drained to the gas-liquid separator, where an argon flow carried the tin vapours into the ICP-AES torch, while the organic eluent was washed out.

Animal Treatment and Sample Preparation

Mussel samples of 4-6 cm in length were exposed to an environment containing TBT in a static sea-water system for several days.1>3,6,9 The mussels were first left in poly- (propylene) tanks in seasonally adjusted, artificial sea-water (15 "C) prepared according to the method of La Roche.22 The 5 ng 1-1 TBT standard solution was added daily, after changing the water. Some mussels were not exposed to the treatment and they were considered as control samples.

Digestive glands, gills and mantle were removed from shells, homogenized and stored at -20 "C until analysis. The procedure for extracting organotin compounds from the tissues consisted of a liquid-liquid extraction, based on the meihod employed by Caricchia et aZ.16 An amount of 3 g of mussel tissue (wet mass) was homogenized using a high-speed homogenizer in 15 ml of a methanol-tropolone mixture (0.05%). The extraction was carried out by sonication and this step was repeated twice. The tissue debris was removed by centrifugation at 3000 rpm for 10 min. The supernatant liquid was collected and 100 ml of de-ionized water and 30 ml of dichloromethane were added. The samples were shaken in a separating funnel for 5 min. The dichloromethane phase, containing the organotins, was collected and evaporated to dryness by using a vacuum rotary evaporator. The samples were redissolved in methanol and 200 p.1 aliquots were injected into the HPLC column. The accuracy of the method was tested by analyses of sub-samples of equal mass of the mussel tissues, spiked with organotin standard solutions.

Results and Discussion

Initial studies were performed to optimize chromatographic separation of standard solutions of TBT, TPT, DBT and

MBT. The effects of the composition of the mobile phase on the separation were examined.

In Fig. 1 the result of a chromatographic separation of a mixture of TBT, TPT and MBT, using 0.1 moll-1 ammonium acetate in 80% viv methanol-water as eluent without the

B

I I 1 0 3 6 9

Retention time/rnin Fig. 1 Chromatographic separation of A, butyltin chloride, B, tributyltin chloride and C, triphenyltin chloride standard solutions. The mobile phase was 0.1 mol 1-1 ammonium acetate in 80% v/v methanol-water .

Table 1 Effect of the presence of tropolone in the eluent on the retention times of organotin compound standard solutions

Compound Without tropolone/min With tropolonelmin Monobutyltin not eluted 3.80 Dibutyltin not eluted 2.70

Triphenyltin 6.70 6.50 Tributyltin 6.11 5.55

100 t B

A

30 I ' I I 0 3 6 9

Fig. 2 Chromatographic separation of A, butyltin chloride, B, dibutyltin chloride and C, tributyltin chloride standard solutions. The mobile phase was 0.1 mol 1-1 ammonium acetate in 80% v/v methanol-water with the addition of 0.1% m/v tropolone.

Retention time/rnin

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Analyst, July 1995, Vol. 120 1939

2o

addition of tropolone is shown. The peaks of the tri-substitutes are asymmetric and MBT only appears when its concentration is high. In fact, to obtain a peak for MBT we had to inject 800 ng of Sn, while for the triorganotin compounds, 40 ng of Sn were injected.

As reported under Experimental, the composition of the mobile phase was modified by adding tropolone (0.1% m/v). Tropolone is often used as a complexing agent in organotin extraction procedures16 and its use as a component of the mobile phase in an HPLC separation of these compounds has also been examined.23 The effects of tropolone on chromato- graphic separation are shown in Table 1 and in Fig. 2. In the presence of this chelating agent, MBT and DBT are eluted; the retention times of the tri-substitutes are not modified significantly, but their peaks are better shaped. The separa- tion is possible within 7 min.

Using these conditions, a detection limit of 7 ng of tin (30) was obtained; this result was two orders of magnitude lower than the values reported in the literature for similar techniques (HPLC-ICP-AES coupling). The LOD was determined by

6 ., . - -.we-- ---.--------.---- -

I I I

I

' Control 3 6 9 Exposure/d

Fig. 4 Concentration levels of A, TBT and B, MBT found in digestive glands of mussels exposed to 5 ng 1- 1 TBT for 3,6 and 9 d and in control animals.

injecting 200 p1 aliquots of standard solutions of different concentrations into the HPLC column.

In order to verify the applicability of the method to real samples, organotin species in tissues of marine mussel exposed to TBT were analysed. The chromatogram of digestive gland extracts of mussels exposed for 9 d to TBT treatment are shown in Fig. 3 Two peaks are present, corresponding to MBT and TBT, respectively.

In Fig. 4, the trends of MBT and TBT found in the digestive glands of mussels exposed to TBT for several days are given.

Conclusion

This method for the analysis of organotin compounds is convenient as it combines the separation facilities of HPLC with the specificity of ICP-AES. The hydride generator, which also works as a gas-liquid separator, allows an increase in the sensitivity of this method and uses organic solvents in high percentages (80%) without extinguishing the plasma torch.

The method is sensitive and reproducible and is not time consuming, allowing a high sample throughput per day. The procedure is suitable for the identification and determination of organotin species in real samples, such as tissues of marine organisms, as organotin compounds are separated and their quantification is possible. Therefore, it may be helpful in monitoring studies assessing the quality of marine environ- ments.

References

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Paper 5/00572H Received January 31, 1995 Accepted March 28, 1995

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