**5. Turbulent-flow chromatography (TFC)**

For a long time the determination of small drug molecules in biological fluids was a very challenging task due to both the complexity of biological samples and the requirement of long chromatographic separations because of the presence of endogenous interferences. The recent implementation of automated on-line extraction procedures has allowed fast sample clean-up in bioanalytical applications, and turbulent-flow (or TurboFlow) chromatography (TFC) appears as one of the most interesting ones in this area [102-105].

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 151

human plasma revealed substantial differences in the overall metabolite profiles compared to methanol-precipitated HPLC-MS, probably due to greatly reduced amounts of phospholipids (ca. 10 fold reduction) with TFC methodology compared to proteinprecipitated samples. TFC seems to be also more efficient at removing proteins based on

**Figure 7.** On-line set-up of a typical TFC-LC-MS system. The position of the various valves to perform the sample clean-up and subsequent analysis of the samples is shown. **a)** Filling the sample loop; **b)** transfer from the sample loop to the TFC column followed by washing; **c)** elution from the TFC column to the Chromatographic column using gradient elution. 1) sample; 2) ASPEC system with a syringe pump; 3) sample loop (10 mL); 4) Waste; 5) HPLC Pump (4 mL min-1); 6) TFC column; 7) Waste; 8)

TFC shows also a big potential in clinical applications, where the increased emphasis on both drug safety and translational biology, e.g. the need to understand how pre-clinical efficacy models are representative of human pharmacology, has considerably modified the expectations for what needs to be measured routinely in biological samples. Moreover, sometimes it is necessary to monitor in the same sample not only the drug levels but also its potential active/reactive metabolites as well as the biomarkers associated with the mechanism of action of the drug. Those biomarkers could span from a very small and polar compounds

HPLC Pump (gradient); 9) HPLC column; 10) MS analyzer. Reproduced from [111].

their size than restricted access media (RAM) or solid phase extraction (SPE) [1,109].

The on-line set-up for a typical TFC-LC-MS method is shown in **Figure 7**. TFC methods are based on the direct injection of biological samples without previous extraction or any treatment into a column packed with large particles. These large particles could have some stationary phase bonded to them, adding an additional selectivity to the extraction procedure. Once the sample have been injected (**Figure 7a**) onto a TurboFlow column, an extraction solution is pumped into the column at a high flow rate (between 1.5 to 5.0 mL min-1) generating turbulent flow conditions inside the column (**Figure 7b**). In general, 100% aqueous mobile buffers are used for this purpose. Under these conditions, small analyte molecules are retained via diffusion processes into the particle pores, while big molecules such as proteins are washed out from the column. In this way, the compounds of interest are extracted from the biological matrix and then eluted from the TurboFlow column onto the analytical column with a volume of solvent which was stored in a holding loop or pumped directly from the chromatographyic LC system (**Figure 7c**). In general organic mobile phases or pH buffered solutions are used for the elution of the compounds of interest depending on the chromatographic separation used after extraction, but the elution volume must be at least ten times that of the TurboFlow column in order to guarantee a complete elution. The analytes are released from the TurboFlow column at a considerably lower flow rate than the one used during extraction into the analytical system where they are mixed with the chromatographic mobile phase and introduced into the chromatographic column, being focused into a sharp band at the head of the HPLC column. When the transfer of analytes is complete, the TurboFlow column could be washed for the next extraction while a regular gradient or isocratic elution is taking place in parallel on the analytical column. The optimization of the different on-line extraction steps is crucial and parameters like mobile phase composition, flow rates and extraction time windows will affect recovery or extraction efficiency in general.

The theory of turbulent flow in open tubes has been discovered and studied for decades. Nevertheless, its application to LC packed columns was only patented in 1997 [106]. The challenge at the moment was to design a chromatographic platform using turbulent flow properties to isolate small analytes from macromolecules present in complex matrices such as biological fluids.

TFC has been used mainly in the handling of biological samples containing a large amount of proteins, such as blood plasma [107,108]. For instance, Michopoulos et al. compared the use of TFC for the metabonomic analysis of human plasma with protein precipitation showing that TFC could be effectively used with the benefit that off-line sample handling was significantly reduced [107]. However, the analysis of the data obtained with TFC for human plasma revealed substantial differences in the overall metabolite profiles compared to methanol-precipitated HPLC-MS, probably due to greatly reduced amounts of phospholipids (ca. 10 fold reduction) with TFC methodology compared to proteinprecipitated samples. TFC seems to be also more efficient at removing proteins based on their size than restricted access media (RAM) or solid phase extraction (SPE) [1,109].

150 Chromatography – The Most Versatile Method of Chemical Analysis

**5. Turbulent-flow chromatography (TFC)** 

extraction efficiency in general.

as biological fluids.

(TFC) appears as one of the most interesting ones in this area [102-105].

For a long time the determination of small drug molecules in biological fluids was a very challenging task due to both the complexity of biological samples and the requirement of long chromatographic separations because of the presence of endogenous interferences. The recent implementation of automated on-line extraction procedures has allowed fast sample clean-up in bioanalytical applications, and turbulent-flow (or TurboFlow) chromatography

The on-line set-up for a typical TFC-LC-MS method is shown in **Figure 7**. TFC methods are based on the direct injection of biological samples without previous extraction or any treatment into a column packed with large particles. These large particles could have some stationary phase bonded to them, adding an additional selectivity to the extraction procedure. Once the sample have been injected (**Figure 7a**) onto a TurboFlow column, an extraction solution is pumped into the column at a high flow rate (between 1.5 to 5.0 mL min-1) generating turbulent flow conditions inside the column (**Figure 7b**). In general, 100% aqueous mobile buffers are used for this purpose. Under these conditions, small analyte molecules are retained via diffusion processes into the particle pores, while big molecules such as proteins are washed out from the column. In this way, the compounds of interest are extracted from the biological matrix and then eluted from the TurboFlow column onto the analytical column with a volume of solvent which was stored in a holding loop or pumped directly from the chromatographyic LC system (**Figure 7c**). In general organic mobile phases or pH buffered solutions are used for the elution of the compounds of interest depending on the chromatographic separation used after extraction, but the elution volume must be at least ten times that of the TurboFlow column in order to guarantee a complete elution. The analytes are released from the TurboFlow column at a considerably lower flow rate than the one used during extraction into the analytical system where they are mixed with the chromatographic mobile phase and introduced into the chromatographic column, being focused into a sharp band at the head of the HPLC column. When the transfer of analytes is complete, the TurboFlow column could be washed for the next extraction while a regular gradient or isocratic elution is taking place in parallel on the analytical column. The optimization of the different on-line extraction steps is crucial and parameters like mobile phase composition, flow rates and extraction time windows will affect recovery or

The theory of turbulent flow in open tubes has been discovered and studied for decades. Nevertheless, its application to LC packed columns was only patented in 1997 [106]. The challenge at the moment was to design a chromatographic platform using turbulent flow properties to isolate small analytes from macromolecules present in complex matrices such

TFC has been used mainly in the handling of biological samples containing a large amount of proteins, such as blood plasma [107,108]. For instance, Michopoulos et al. compared the use of TFC for the metabonomic analysis of human plasma with protein precipitation showing that TFC could be effectively used with the benefit that off-line sample handling was significantly reduced [107]. However, the analysis of the data obtained with TFC for

**Figure 7.** On-line set-up of a typical TFC-LC-MS system. The position of the various valves to perform the sample clean-up and subsequent analysis of the samples is shown. **a)** Filling the sample loop; **b)** transfer from the sample loop to the TFC column followed by washing; **c)** elution from the TFC column to the Chromatographic column using gradient elution. 1) sample; 2) ASPEC system with a syringe pump; 3) sample loop (10 mL); 4) Waste; 5) HPLC Pump (4 mL min-1); 6) TFC column; 7) Waste; 8) HPLC Pump (gradient); 9) HPLC column; 10) MS analyzer. Reproduced from [111].

TFC shows also a big potential in clinical applications, where the increased emphasis on both drug safety and translational biology, e.g. the need to understand how pre-clinical efficacy models are representative of human pharmacology, has considerably modified the expectations for what needs to be measured routinely in biological samples. Moreover, sometimes it is necessary to monitor in the same sample not only the drug levels but also its potential active/reactive metabolites as well as the biomarkers associated with the mechanism of action of the drug. Those biomarkers could span from a very small and polar compounds

such as a neurotransmitter to a very large and hydrophobic entity like fatty acids. So, amongst the key analytical challenges with biomarkers is generally the sampling procedure as well as the sample volume available. TFC has some intrinsic capabilities that facilitate the analysis of biomarkers and metabolites [103]. First, it provides high sensitivity assays without the need for high sample volume. In addition, the on-line extraction approach removes the need for lengthy sample preparation procedures, hence reducing sample degradation issues frequently observed in biomarker analysis. As an example, Mueller et al. proposed a fully automated toxicological LC-MSn screening system in urine using on-line extraction with TFC [110].

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 153

In a recently published review dedicated to sample preparation methodologies for the isolation of veterinary drugs and growth promoters from food, Kinsella et al. described turbulent flow chromatography as a technique that eliminates time-consuming sample clean-up, increases productivity and reduces solvent consumption without sacrificing sensitivity [116]. Food matrices have a high content of fat and proteins, which makes TFC an ideal sample treatment technique for the determination of a specific class of contaminants in various matrices such as honey, tissues and milk [117]. Some examples are described in the literature concerning the determination of veterinary drugs such as quinolones in honey and animal tissue [13-14]. For instance, turbulent flow chromatography coupled to LC-MS/MS was proposed for the quantitative high-troughput analysis of 4 quinolones and 12 fluoroquinolones in honey [13]. The manual sample preparation was limited to a simple dilution of the honey test portion with water followed by a filtration. The extract was then on-line purified on a large particle size TFC column where the sample matrix was washed away while the analytes were retained. Recoveries of 85-127% were obtained, while matrix effects were still observed which led to the use of standard addition for calibration. The proposed methodology has also shown good robustness, with over 400 injections of honey extracts without any TFC column deterioration, with the consumption of 44 mL of solvent per sample. The authors described that TFC showed a strong potential as an alternative extraction and clean-up sample method compared to those making use of off-line sample preparation, in terms of both increasing the analysis throughput and obtaining higher reproducibility linked to automation to ensure the absence of contaminants in honey samples. In the case of animal tissues TFC was used for sample preparation in the analysis of two quinolones (enrofloxacin and its metabolite ciprofloxacin) [14]. Sample was extracted with a mixture of acetonitrile/water 1:1 acidified with 0.01% formic acid. Mean recovery rates for the tissues of the different species (cattle, pig, turkey and rabbit) were in the range

Presta et al. [111] described the use of TFC coupled to LC-MS for the determination of flavonoids and resveratrol in wines. 10 mL of sample (diluted wine) was passed over the TFC column, after which the retained analytes were separated by reversed-phase LC. The

Turboflow chromatography has also been described for sample treatment in the screening of eight veterinary drugs in milk [15]. Protein precipitation was induced before analyzing samples of whole, skimmed and semi-skimmed milk samples. While matrix effects – ion suppression and enhancement – were obtained for all analytes, the method has proved to be useful for screening purposes because of its sensitivity (0.1 to 5.2 µg L-1), linearity and repeatability (RSD ≤ 12%). As an example, **Figure 8** shows the chromatographic separation of a non-fat milk sample spiked with target veterinary drugs and analyzed by TFC-LC-(ESI)-

This sample treatment technique has also been applied successfully to environmental samples. For instance, anti-infectives analysis in wastewater has been reported with good recovery (86-141%) and limits of quantification (45-122 ng L-1) [114]. Signal distortion,

of 72-105% in a run time of only 4 min.

MS/MS.

method proved to be fast, non-laborious, robust and sensitive.

Very recently the use of TFC couple to tandem mass spectrometry has been reported for the automated analysis of perfluorinated compounds (PFCs) in human hair and urine samples [112]. The method allowed the extraction and analysis of 21 PFCs with recoveries between 60 to 105%.

But today, TFC is being used in other fields of applications, such as food or even environmental analysis. **Table 4** shows some examples of these TFC applications.


**Table 4.** Relevant examples of the application of turbulent flow chromatography.

In a recently published review dedicated to sample preparation methodologies for the isolation of veterinary drugs and growth promoters from food, Kinsella et al. described turbulent flow chromatography as a technique that eliminates time-consuming sample clean-up, increases productivity and reduces solvent consumption without sacrificing sensitivity [116]. Food matrices have a high content of fat and proteins, which makes TFC an ideal sample treatment technique for the determination of a specific class of contaminants in various matrices such as honey, tissues and milk [117]. Some examples are described in the literature concerning the determination of veterinary drugs such as quinolones in honey and animal tissue [13-14]. For instance, turbulent flow chromatography coupled to LC-MS/MS was proposed for the quantitative high-troughput analysis of 4 quinolones and 12 fluoroquinolones in honey [13]. The manual sample preparation was limited to a simple dilution of the honey test portion with water followed by a filtration. The extract was then on-line purified on a large particle size TFC column where the sample matrix was washed away while the analytes were retained. Recoveries of 85-127% were obtained, while matrix effects were still observed which led to the use of standard addition for calibration. The proposed methodology has also shown good robustness, with over 400 injections of honey extracts without any TFC column deterioration, with the consumption of 44 mL of solvent per sample. The authors described that TFC showed a strong potential as an alternative extraction and clean-up sample method compared to those making use of off-line sample preparation, in terms of both increasing the analysis throughput and obtaining higher reproducibility linked to automation to ensure the absence of contaminants in honey samples. In the case of animal tissues TFC was used for sample preparation in the analysis of two quinolones (enrofloxacin and its metabolite ciprofloxacin) [14]. Sample was extracted with a mixture of acetonitrile/water 1:1 acidified with 0.01% formic acid. Mean recovery rates for the tissues of the different species (cattle, pig, turkey and rabbit) were in the range of 72-105% in a run time of only 4 min.

152 Chromatography – The Most Versatile Method of Chemical Analysis

**Compounds Sample TFC column** 

Edible tissues (cattle, pig, turkey, rabbit)

Wine

Surface, drinking wáter

PFOS River water

infectives Wastewater

60 to 105%.

Quinolones Honey

Quinolones (Enrofloxacin

Ciprofloxacin)

drugs Milk

Veterinary

Flavonoids and resveratrol

Anti-

Pesticides

and

such as a neurotransmitter to a very large and hydrophobic entity like fatty acids. So, amongst the key analytical challenges with biomarkers is generally the sampling procedure as well as the sample volume available. TFC has some intrinsic capabilities that facilitate the analysis of biomarkers and metabolites [103]. First, it provides high sensitivity assays without the need for high sample volume. In addition, the on-line extraction approach removes the need for lengthy sample preparation procedures, hence reducing sample degradation issues frequently observed in biomarker analysis. As an example, Mueller et al. proposed a fully automated toxicological LC-MSn screening system in urine using on-line extraction with TFC [110].

Very recently the use of TFC couple to tandem mass spectrometry has been reported for the automated analysis of perfluorinated compounds (PFCs) in human hair and urine samples [112]. The method allowed the extraction and analysis of 21 PFCs with recoveries between

But today, TFC is being used in other fields of applications, such as food or even

**Flow-rate / Injection volume** 

1.5 mL min-1 /

5 mL min-1 /

1.5 mL min-1 /

4 mL min-1 / 10 mL

1 mL min-1 /

3 mL min-1 /

5 mL min-1 / 10 mL

**Detection Reference** 

160 µL LC-ESI-MS/MS [13]

20 µL LC-ESI-MS/MS [14]

50 µL LC-ESI-MS/MS [15]

LC-ESI-MS

1 mL LC-APPI-MS [113]

1 mL LC-ESI-MS/MS [114]

LC-APPI-

LC-APCI-MS [111]

MS/MS [115]

environmental analysis. **Table 4** shows some examples of these TFC applications.

Cyclone HTLC, 50 x 0.5 mm, 60 µm (Thermo Fisher Scientific)

Cyclone HTLC, 50 x 1.0 mm, 50 µm (Thermo Fisher Scientific)

Cyclone – Cyclone P connected in tandem, 50 x 0.5 mm, 60 µm (Thermo Fisher Scientific)

50 x 1.0 mm, 60 µm C18 (Thermo Fisher

50 x 1.0 mm, 50 µm C18 (Cohesive Technologies)

50 x 1.0 mm, 50 µm C18 SL (Cohesive Technologies)

50 x 1.0 mm, 35 µm

Oasis HLB (Waters)

**Table 4.** Relevant examples of the application of turbulent flow chromatography.

Scientific)

Presta et al. [111] described the use of TFC coupled to LC-MS for the determination of flavonoids and resveratrol in wines. 10 mL of sample (diluted wine) was passed over the TFC column, after which the retained analytes were separated by reversed-phase LC. The method proved to be fast, non-laborious, robust and sensitive.

Turboflow chromatography has also been described for sample treatment in the screening of eight veterinary drugs in milk [15]. Protein precipitation was induced before analyzing samples of whole, skimmed and semi-skimmed milk samples. While matrix effects – ion suppression and enhancement – were obtained for all analytes, the method has proved to be useful for screening purposes because of its sensitivity (0.1 to 5.2 µg L-1), linearity and repeatability (RSD ≤ 12%). As an example, **Figure 8** shows the chromatographic separation of a non-fat milk sample spiked with target veterinary drugs and analyzed by TFC-LC-(ESI)- MS/MS.

This sample treatment technique has also been applied successfully to environmental samples. For instance, anti-infectives analysis in wastewater has been reported with good recovery (86-141%) and limits of quantification (45-122 ng L-1) [114]. Signal distortion, represented as matrix effect, was still observed probably due to the fact that small molecules (below 1000 Da) present in wastewater samples will have affinity for the stationary phase and will not be completely removed in the clean-up step. Takino et al. have minimized the matrix effect observed by using atmospheric pressure photoionization (APPI) instead of electrospray (ESI) as ionization source [113]. In this case, a simple, fast and sensitive LC/APPI-MS method, with automated on-line extraction using TFC was developed for the determination of perfluorooctane sulfonate (PFOS) in river water. TFC columns packed with organic polymers or graphitized carbons were also found to be highly capable for enrichment of trace pesticides from drinking and surface water samples [115].

Current Trends in Sample Treatment Techniques for Environmental and Food Analysis 155

useful method in the area of food analysis, especially in matrices with a high content of fat

There is an increasing demand for high-throughput chromatographic separations in food and environmental analysis where highly heterogeneous and difficult matrices may be analyzed. Despite the important advances in chromatographic separations, food and environmental matrices are very complex samples, and sample extraction and clean-up treatments are usually required. Therefore, sample treatment is still one of the most important parts of whole analytical method and effective sample preparation is crucial in achieving accurate analytical results. Food and environmental analysis generally requires several steps such as extraction from the sample of interest, removal of co-extracted matrix components, analytes enrichment and their subsequent quantification. Thus, the availability of robust, sensitive, selective and rapid analysis methods is of primary importance. The most recently introduced sample treatment methodologies in food and environmental applications have been discussed in this chapter, such as on-line SPE methods, QuEChERS,

MIPs as selective sorbents for SPE, and the use of turbulent-flow chromatography.

associated with the classical off-line SPE procedure.

great potential for the analysis of protein- and fat-rich matrices.

i.e., high specificity, selectivity and capacity.

chlorinated compounds.

On-line SPE is a viable and increasingly popular technique used to improve the sample throughput by reducing sample preparation time and overcome many of the limitations

For sensitive and selective determination of compounds in very complex matrices, the use of polymers with recognition sites able to specifically bind a particular substance or a group of structural analogues has attracted increase attention due to their outstanding advantages,

QuEChERS appeared as satisfactory, simple, rapid and inexpensive sample extraction and clean-up multi-residue methods especially employed in the analysis of pesticides. However, this methodology is also being successfully employed for the extraction of other families of compounds in food and environmental matrices such as acrylamide, mycotoxins, PAHs and

The use of turbulent-flow chromatography represents a highly attractive and promising approach for removing proteins based on their size better than RAM or SPE procedures. TFC has been satisfactory applied to the direct analysis of complex matrices with reduced or without any sample manipulation and, even not many applications in food and environmental samples are yet available, it will become a very useful method due to its

Finally, future developments in all areas of analytical sample preparation are expected to continue in order to improve accuracy, sensitivity, specificity, and reproducibility of the sample treatment technique together with reduced analysis time and sample manipulation.

and proteins such as milk.

**6. Conclusion** 

**Figure 8.** Representative SRM chromatograms of a non-fat milk sample spiked with the mixture of antibiotics standards at 100 µg L-1 level and analyzed by TFC-LC-(ESI)-MS/MS. Reproduced from [15] with permission from Springer.

In summary, turbulent flow chromatography appears as a very useful approach for sample treatment because it possesses greater efficiency in removing proteins based on their size than restricted access media or SPE procedures and combines high-throughput and high reproducibility by means of separating analytes from various matrices with reduced sample handling. The advantages of this sample extraction and clean-up procedure is unquestionable in bioanalytical applications, and although not many applications in other fields such as food and environmental analysis are yet available, it will surely become a very useful method in the area of food analysis, especially in matrices with a high content of fat and proteins such as milk.

## **6. Conclusion**

154 Chromatography – The Most Versatile Method of Chemical Analysis

water samples [115].

with permission from Springer.

represented as matrix effect, was still observed probably due to the fact that small molecules (below 1000 Da) present in wastewater samples will have affinity for the stationary phase and will not be completely removed in the clean-up step. Takino et al. have minimized the matrix effect observed by using atmospheric pressure photoionization (APPI) instead of electrospray (ESI) as ionization source [113]. In this case, a simple, fast and sensitive LC/APPI-MS method, with automated on-line extraction using TFC was developed for the determination of perfluorooctane sulfonate (PFOS) in river water. TFC columns packed with organic polymers or graphitized carbons were also found to be highly capable for enrichment of trace pesticides from drinking and surface

**Figure 8.** Representative SRM chromatograms of a non-fat milk sample spiked with the mixture of antibiotics standards at 100 µg L-1 level and analyzed by TFC-LC-(ESI)-MS/MS. Reproduced from [15]

In summary, turbulent flow chromatography appears as a very useful approach for sample treatment because it possesses greater efficiency in removing proteins based on their size than restricted access media or SPE procedures and combines high-throughput and high reproducibility by means of separating analytes from various matrices with reduced sample handling. The advantages of this sample extraction and clean-up procedure is unquestionable in bioanalytical applications, and although not many applications in other fields such as food and environmental analysis are yet available, it will surely become a very There is an increasing demand for high-throughput chromatographic separations in food and environmental analysis where highly heterogeneous and difficult matrices may be analyzed. Despite the important advances in chromatographic separations, food and environmental matrices are very complex samples, and sample extraction and clean-up treatments are usually required. Therefore, sample treatment is still one of the most important parts of whole analytical method and effective sample preparation is crucial in achieving accurate analytical results. Food and environmental analysis generally requires several steps such as extraction from the sample of interest, removal of co-extracted matrix components, analytes enrichment and their subsequent quantification. Thus, the availability of robust, sensitive, selective and rapid analysis methods is of primary importance. The most recently introduced sample treatment methodologies in food and environmental applications have been discussed in this chapter, such as on-line SPE methods, QuEChERS, MIPs as selective sorbents for SPE, and the use of turbulent-flow chromatography.

On-line SPE is a viable and increasingly popular technique used to improve the sample throughput by reducing sample preparation time and overcome many of the limitations associated with the classical off-line SPE procedure.

For sensitive and selective determination of compounds in very complex matrices, the use of polymers with recognition sites able to specifically bind a particular substance or a group of structural analogues has attracted increase attention due to their outstanding advantages, i.e., high specificity, selectivity and capacity.

QuEChERS appeared as satisfactory, simple, rapid and inexpensive sample extraction and clean-up multi-residue methods especially employed in the analysis of pesticides. However, this methodology is also being successfully employed for the extraction of other families of compounds in food and environmental matrices such as acrylamide, mycotoxins, PAHs and chlorinated compounds.

The use of turbulent-flow chromatography represents a highly attractive and promising approach for removing proteins based on their size better than RAM or SPE procedures. TFC has been satisfactory applied to the direct analysis of complex matrices with reduced or without any sample manipulation and, even not many applications in food and environmental samples are yet available, it will become a very useful method due to its great potential for the analysis of protein- and fat-rich matrices.

Finally, future developments in all areas of analytical sample preparation are expected to continue in order to improve accuracy, sensitivity, specificity, and reproducibility of the sample treatment technique together with reduced analysis time and sample manipulation.
