Abstract

Miniaturized separation techniques have emerged as environmentally friendly alternatives to available separation methods. Nano-liquid chromatography (nano-LC), microchip devices and nano-capillary electrophoresis are miniaturized methods that minimize reagent consumption and waste generation. Furthermore, the low levels of analytes, especially in biological samples, promote the search for more highly sensitive techniques; coupled to mass spectrometry, nano-LC has great potential to become an indispensable tool for routine analysis of biomolecules. This short review presents the fundamental aspects of nano-LC analytical instrumentation, discussing practical considerations and the primary differences between miniaturized and conventional instrumentation. Some theoretical aspects are discussed to better explain both the potential and the principal limitations of nano-LC. Recent pharmaceutical and biomedical applications of this separation technique are also presented to indicate the satisfactory performance for complex matrices, especially for proteomic analysis, that is obtained with nano-LC.

Introduction

The development of miniaturized systems is not a recent event. In the 1950s, the use of capillary columns for gas chromatography was proposed by Golay (1), and Horváth et al. used columns with small internal diameters (i.d.) for liquid chromatography (LC) separations in the ‘60s (2). The nano-LC technique, as it is currently known, was first introduced by Karlsson and Novotny in 1988 (3), testing packed columns with very small i.d.

Recently, the great increase in miniaturized LC systems has been driven by biological applications, primarily proteomics research. Mixtures of proteins or peptides must be analyzed, and the quantities of available samples and the low concentrations of the target analytes in the sample matrix are not compatible with conventional LC systems.

Traditional analyses by high-performance liquid chromatography (HPLC) are performed using columns with i.d. of 3.5–4.6 mm. These analytical columns have typical flow rates of 1.0 mL/min. Columns with smaller dimensions (internal diameters of 20–100 µm) that use flow rates of nanoliters per minute are called nano columns (4) and are used in nano-LC.

Nano-LC is an alternative to conventional LC, providing more options for chemical analysis. Virtually all samples analyzed by conventional LC can be analyzed by a miniaturized technique. In this context, capillary electrophoresis and capillary electrochromatography also complement and compete with nano-LC as miniaturized liquid phase separations.

Developments in nano-LC are tied to the various advantages offered by this technique over conventional HPLC analyses. Some positive aspects are: (i) the large decrease in mobile and stationary phase consumption, including toxic reagents; (ii) the small sample needed; (iii) the high efficiency separations while maintaining the same retention behavior; (iv) the easy coupling to mass spectrometry (MS). Currently, one of the most important advantages is reduced waste generation, in accordance with the principles of green chemistry (5–7).

However, the analytical instrumentation used in nano-LC is still very expensive, limiting its widespread use. Moreover, significant technical knowledge about nano-LC details is required to prevent experimental difficulties, especially those related to the instrumental arrangement.

The number of publications focusing on the applications of nano-LC has grown in recent years; however, neither the theoretical aspects nor those related to instrumentation are reported in these publications. Thus, this review covers the principal aspects of the nano-LC technique and some recent pharmaceutical and biomedical applications, especially in the most employed area, biological research. Proteomic analysis, which corresponds to the major application of nano-LC, is also presented.

Principles of Nano-LC

There is no agreement about the terminology of microscale LC. The terms “microbore,” “microcolumn” and “capillary” LC are used interchangeably for microcolumns of different i.d. (8). According to Chervet et al. (9), separations performed using columns of 0.50–1.0 mm i.d. are described as micro-LC; columns of 100–500 µm i.d. are described as capillary-LC; finally, separations using columns of 10–100 µm i.d. are described as nano-LC. This classification includes separations in microchips because nano-HPLC columns on chips have 20 to 100 µm as the i.d.

In this work, the classification of nano-LC will be used for separations that operate at nanoliter flow rates, common for columns of 10–100 µm i.d.

Theoretical aspects of nano-LC

During the chromatographic process, injected analytes can undergo dilution in the column that alters separation efficiency. This dilution event, called chromatographic dilution (D), is expressed by  

(1)
formula
where Co is the initial concentration and Cmax is the final concentration of the analyte during the chromatographic process, dc is the column internal diameter, ε is the total porosity of the column, L is the column length, Vinj is the sample injection volume and k and H are the chromatographic parameters retention factor and plate height, respectively.

According to the equation, D decreases proportionally with the reduction of the square of the column diameter. Compared to conventional HPLC, the lower i.d. in nano-LC promotes a high reduction in D value. Thus, downscaling of chromatographic systems means less chromatographic dilution, increasing the mass detectability of the separation (6).

The flow rate (F) in a column is given by  

(2)
formula
where u is the linear velocity of the mobile phase.

The reduction of dc leads to a large reduction in the flow rate of the mobile phase, decreasing solvent consumption and waste production in nano-LC separations.

Theoretically, the miniaturization of LC systems is very advantageous for liquid phase separations. However, some practical separation aspects must be considered, because they contribute to losses in separation efficiency.

Analytical instrumentation of nano-LC systems

In nano-LC, the conventional instrumentation is all miniaturized. Pumps, connections, columns, injection loop and detection interface are dimensioned for small volumes and low back-pressure. These parameters can greatly influence the chromatographic efficiency of nano-LC and need to be controlled for a successful separation.

Pumps

Pumps for nano-LC need to present reproducible nano flow rates and stability during the separation, and permit gradient elution at nano-scale levels. Two primary systems can be used in nano-LC: split and splitless pumps, the latter being commercially available.

Split systems divide high flows (µL/min) from conventional HPLC pumps by using a flow restrictor between the pump and the miniaturized column. These systems allow for the use of the usual HPLC pumps with an easily constructed nano flow restrictor (10). However, split systems may lead to variable split ratios and low reproducibility of the nano flow, decreasing the repeatability of the separation (10). Reproducible gradient elution is very difficult to achieve, especially with homemade splitting devices: the different viscosities of the mixed solvents can cause back-pressure fluctuations, limiting this elution mode (11).

Currently, splitless systems are widely used in nano-LC. These systems prevent solvent losses and have more reproducible nano flow rates. Syringe pumps using a single reservoir with a limited volume are better than split systems, but continuous flow pumps, similar to conventional reciprocating pumps with two pistons per channel, are currently the most widely used pump model. Continuous flow pumps can be used in both isocratic and gradient elution at nano flows and adjustments of the desired nano flow rates are easily achieved.

Tubing and connections

Peak broadening is a significant limitation to nano-LC development. The dispersion of an analyte band in the column is described as a function of the i.d. and length of the capillary (7, 12).

Thus, the lower the i.d. and length, the lower the on-column dispersion contribution (12). Pre-column and post-column dead volumes may lead to significant band broadening, which is critical when using columns with reduced i.d. Inadequate tubing and connections increase band broadening, so that the use of short, tight connections made with low volume tubing is required to reduce this contribution to band broadening (7). Common connections are made of stainless steel or polyetheretherketone; (PEEK) the latter is especially useful for fused silica capillaries.

Spaces formed by inadequate connections can also promote lower separation resolution. Noga et al. (13) reviewed some common limitations to nano-LC separations, including problems with connections and their practical resolution. They performed separations of bovine serum albumin (BSA) digests, comparing both inadequate and adequate connections. Figure 1 shows the effect of dead volume on the quality of the chromatographic separations. According to the authors, inadequate fittings in the system cause a severe mixing inside the system and no peaks were detected until 35 min2.

Figure 1.

Separation of BSA digest using a C18 packed column (75 µm i.d. × 5 cm) at a flow rate of 300 nL/min with MS detection (range 400–1,400 m/z). The top frame shows the analysis with dead volume in the pre-column connections, whereas the bottom frame shows the same analysis repeated after the problem is solved. Reprinted from Journal of Separation Science, Vol 30, Author(s): Noga, M., Sucharski, F., Suder, P., Silberring, J., A practical guide to nano-LC troubleshooting, Pages 2179-2189, Copyright (2007) with permission from John Wiley and Sons.

Figure 1.

Separation of BSA digest using a C18 packed column (75 µm i.d. × 5 cm) at a flow rate of 300 nL/min with MS detection (range 400–1,400 m/z). The top frame shows the analysis with dead volume in the pre-column connections, whereas the bottom frame shows the same analysis repeated after the problem is solved. Reprinted from Journal of Separation Science, Vol 30, Author(s): Noga, M., Sucharski, F., Suder, P., Silberring, J., A practical guide to nano-LC troubleshooting, Pages 2179-2189, Copyright (2007) with permission from John Wiley and Sons.

Figure 2.

Extracted ion chromatograms of BSA digest analyzed on different capillary columns: 3 µm porous particles (a); 2.7 µm fused-core particles (b); silica monolith (c); polymeric monolith (d). Separation conditions: flow rate of 1,000 nL/min, gradient time of 7.5 min. Injected amounts of sample: 500 fmol for silica-based columns and 200 fmol for the polymeric column MS detection. Reprinted from Journal of Chromatography B, Vol 912, Author(s): Dolman, S., Eeltink, S., Vaast, A., Pelzing, M., Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns, Pages 56-63, Copyright (2012) with permission from Elsevier.

Figure 2.

Extracted ion chromatograms of BSA digest analyzed on different capillary columns: 3 µm porous particles (a); 2.7 µm fused-core particles (b); silica monolith (c); polymeric monolith (d). Separation conditions: flow rate of 1,000 nL/min, gradient time of 7.5 min. Injected amounts of sample: 500 fmol for silica-based columns and 200 fmol for the polymeric column MS detection. Reprinted from Journal of Chromatography B, Vol 912, Author(s): Dolman, S., Eeltink, S., Vaast, A., Pelzing, M., Investigation of carryover of peptides in nano-liquid chromatography/mass spectrometry using packed and monolithic capillary columns, Pages 56-63, Copyright (2012) with permission from Elsevier.

Injection

The maximum injection volumes for nano columns can be expressed as a function of the column length, plate number, retention factor or some other parameters, and are generally a few nanoliters (9). Small injected volumes are a major problem in nano-LC, causing loss of detectability, but larger injected volumes produce a band broadening effect, decreasing the efficiency of the separation, especially for poorly retained compounds. However, Heron et al. proved that, when using a weak solvent for the sample, there is an enrichment effect and a gain in efficiency, promoting the concentration of a sample plug after injection into a stronger mobile phase (14).

Commercial autosamplers, which usually work at microliter levels, require an instrument adjustment for use in the nanoliter range. This may be overcome by the use of a split valve between the injector and the column (9).

Nano-columns

Although columns of 10 µm i.d. can be employed, nano-LC columns of 75 µm i.d. are the most frequently used. This i.d. column provides a good compromise between detectability, loadability and robustness in nano-LC separations (10).

In general, nano-LC columns are made of polyimide-coated fused silica capillaries that present flexibility, high mechanical resistance and a variety of internal dimensions, but stainless steel and titanium tubes are also used for nano columns. They can be packed with silica-based particles, filled by a monolithic bed or, less commonly, wall-coated with appropriate organic or inorganic materials.

The most common particle sizes for packed nano columns are 3–5 µm. However, particle-filled small i.d. columns are difficult to prepare. Retention frits are required to prevent the stationary phase from escaping; the preparation of the frits also presents low reproducibility and often results in decreased efficiencies (15). Nonhomogeneous beds after packing also reduce chromatographic performance (15).

Monolithic stationary phases are single rods of organic or inorganic material that are produced inside the capillary column. No frits are required with monolithic columns and the high porosity of these materials allows higher flow rates of mobile phase, reducing the separation time (16). Monoliths can be prepared by using different synthesis routes, organic or inorganic-based, and biocompatible materials are interesting alternatives in biospecific analyses (17).

Dolman et al. (18) compared the performances of packed and monolithic stationary phases for BSA separations and evaluated the carryover effects in different capillary columns (Figure 2). BSA was chosen as a model for typical proteomic samples because it contains both hydrophilic and hydrophobic peptides. Better separation efficiencies of the 11 peptides were attributed to the silica monolithic column, very similar to the efficiency of the fused-core 2.7 µm silica packed column. The carryover effect, which can compromise peptide analyses, was less with the fused-core 2.7 µm silica than with the polymeric monolith and the porous 3 µm silica packed columns also employed by the authors.

The chemistry available for stationary phases allows the applicability of nano-LC in a range of analyses. Reversed-phase, hydrophilic interaction chromatography (HILIC), chiral selection, size exclusion, ion exchange and other separation modes are applied to separations, according to the target analytes. Many research groups prefer to prepare their own nano columns specifically for their own purposes.

A chiral stationary phase for nano-LC was developed by Fanali et al. (19). In this work, cellulose tris(3-chloro-4-methylphenylcarbamate)-coated silica particles were employed for the separation of six neutral drugs, including thalidomide, a teratogenic drug. Enantiomeric separations using a column of 100 µm i.d. were achieved in under 10 min and were especially good for the separation of thalidomide, which cannot be commercialized as a racemic mixture.

Detection

The types of detection for nano-LC are the same as those employed for HPLC separations. Diode array detection (DAD) is commonly used in nano-LC, because of its low cost, wide range of applicability and use of online detection. However, due to the short path length of the nano column, detectability is limited when on-column detection is applied. This is overcome by the use of specially configured detection cells that provide longer light paths (11). Laser induced fluorescence (20) and inductively coupled plasma MS (21) are also used in nano-LC detection, but these are not robust enough to be applied for routine analysis.

Biomedical and pharmaceutical applications usually require good detectability and a universal detection method, such as that provided by MS detection. The nano flow from the column (frequently, 100–500 nL/min) is adequate for MS coupling through various nanospray interfaces, especially electrospray ionization (ESI), which requires only a small amount of eluent from the LC column to be successful (22).

Enrichment in nano-LC

Theoretically, the use of nano-LC promotes analyte enrichment more than conventional HPLC. Reduced i.d. columns decrease chromatographic dilution and, consequently, increase the instantaneous concentration of the injected analyte as it passes through the components of the LC instrument. This enrichment factor is attributed to lower dilution factors and is proportional to the square of the column radius and the injected analyte volume, as discussed by Rieux et al. (10). The smaller the column radius, the lower the dilution factor, with a resulting increase in analyte detectability.

According to Cutillas (4), the use of a column of 75 µm i.d. can lead to an analyte concentration factor of 5,000, compared to the use of a column of 4.6 mm i.d.. However, this enrichment factor is not easily achieved, because other instrumental factors often decrease the observed analyte concentration, such as excessive connection tubing, dead volumes from connections and disrupted nano flow.

Some experimental observations show that the small sample volumes injected decrease the detectability of nano-LC compared to conventional HPLC, especially when using ultraviolet (UV) detection (11). The application of MS detection, multidimensional (nano)-LC or on-column trapping can greatly increase detectability in nano-LC.

Hyphenation in nano-LC

MS is the most common nano-LC hyphenation. Coupling nano-LC to MS or tandem mass spectrometry (MS-MS) has been applied in different areas, which has solved various problems in the analytical sciences. For example, nano-LC separations coupled to online MS (or, less commonly, offline MS) have increased the diagnosis and treatment of several human diseases, promoting better quality of life (23–25).

Nano columns are also suitable for coupling to secondary separation techniques, resulting in a two-dimensional (2D) chromatographic system. Luo et al. (26) proposed the 2D separation of a complex proteomic analysis from cervical cancer cells using strong cation exchange and reversed-phase wall-coated nano columns, coupled to MS detection. The authors concluded that the separation capacity was improved by the orthogonality of the open tubular columns, compared to one-dimensional reversed-phase.

Hyphenations also can be applied by using two miniaturized schemes, such as biological microanalysis systems (27) or coupling another (orthogonal) nano column in the second dimension of the 2D separation (26, 28). However, few reports of this kind of hyphenation have been published to date, probably due to instrumental limitations.

Recent Pharmaceutical and Biomedical Applications of Nano-LC

Molecules of biological interest have to be quickly determined with highly reliable results. In this context, recent advances in analytical instrumentation and sample preparation methods have propelled biological analyses for the identification of these interesting molecules.

Nano-LC analyses are now applied for therapeutic and veterinary drugs, doping control, disease diagnosis and the quantitative determination of biomarkers and proteome identification; the latter are the principal application fields, primarily because of the very low sample amount required.

Proteomic research

Undoubtedly, proteomic studies respond to the major application of nano-LC separations (29–34). Protein sequencing of complex biological samples is necessary for biomarker identifications, disease control and clinical treatments, principally from plasma and tissue samples.

HPLC-based methods overcome the classical problems of protein analysis, such as gel electrophoresis and immunoanalysis, which are both limited by multiple steps before analyses. The diversity of proteome complexity requires fast and unquestionable identification techniques, promoted by the emergence of nano-LC coupled to MS and MS-MS. These have allowed the exact determination of amino acid sequences from proteins or peptides, which is assisted by a full identification database. However, classical methods are still used with nano-LC–MS, because much information about protein sequencing and peptide mapping is obtained by a combination of two or more identification strategies.

If not correctly diagnosed and treated, periodontitis can lead to acute loss of teeth and systemic complications. Choi et al. (35) proposed the identification of the proteins of gingival samples from healthy and periodontitic patients while searching for a specific biomarker for this inflammatory disease. The authors used nano-LC–MS-MS for proteomic analysis and immunoassays for test confirmation; 305 proteins were identified in both sick and healthy patients and among these, 45 were directly related to periodontitis. Azurocidin was chosen as the best biomarker and its levels were highly augmented in periodontitis patients, inhibiting osseous differentiation in these cases. The principal conclusion of this work was to propose the early diagnosis of periodontitis by the direct measurement of azurocidin levels by nano-LC–MS-MS in complex oral samples, preventing the complications from untreated disease.

Proteomic analyses have been performed for synovial fluid from rheumatic patients by using nano-LC–MS-MS (36). Osteoarthritis and rheumatoid arthritis are both destructive articular diseases, characterized by a gradual degradation of the cartilage tissues by defense cells, followed by inflammation disturbances. Mateos et al. (36) identified peptides related to both articular diseases and other peptides exclusive to each one. Knowledge of the proteome from synovial fluids was important to detect protein fractions that acted as biomarkers and promoted an efficient clinical control of patient treatments.

Table I lists other proteomic analyses conducted by nano-LC that have been reported in the last two years (28, 37–43).

Table I

Recent Proteomic and Peptide Analyses Using Nano-LC Techniques Coupled to MS Detection*

Matrix Proteome Nano-LC conditions Reference 
Human embryos Proteins secreted by pre-implantation embryos 750 ng digested sample injected; C18 (1.7 µm, 100 µm i.d. × 10 cm); ACN gradient, 600 nL/min; TOF analyzer 28 
Rice roots (O. sativa L.) Ubiquitinated proteins after salt stress 5 µL digested peptide injected; C18 (5 µm, 75 µm i.d. × 11 cm); ACN gradient, 250 nL/min; ion trap analyzer 37 
Cheese Enzymes produced during ripening 10 µL hydrolyzed sample injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 50 nL/min; both MALDI and ESI; Q-TOF analyzer 38 
Marine shells Proteins from layers of the shell 1 µL digested sample injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 50 nL/min; Q-TOF analyzer 39 
Saccharomyces cerevisae Proteins from whole yeast cell 2 µL digested sample injected; C18 (5 µm, 75 µm i.d. × 15 cm); ACN gradient, 350 nL/min; ion trap analyzer 40 
Human corneas Proteins from healthy and keratoconus corneas 5 µL extracted tissue injected; C18 (5 µm, 100 µm i.d. × 11 cm); ACN gradient, 500 nL/min; ion trap analyzer 41 
Preclinical mouse models Monoclonal antibodies from mouse plasma 2 µL treated sample injected; C18 (5 µm, 75 µm i.d. × 25 cm); ACN gradient, 250 nL/min; ion trap analyzer 42 
Candida albicans Surface proteins from cell wall 108 digested cells injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 300 nL/min; TOF analyzer after UV detection 43 
Matrix Proteome Nano-LC conditions Reference 
Human embryos Proteins secreted by pre-implantation embryos 750 ng digested sample injected; C18 (1.7 µm, 100 µm i.d. × 10 cm); ACN gradient, 600 nL/min; TOF analyzer 28 
Rice roots (O. sativa L.) Ubiquitinated proteins after salt stress 5 µL digested peptide injected; C18 (5 µm, 75 µm i.d. × 11 cm); ACN gradient, 250 nL/min; ion trap analyzer 37 
Cheese Enzymes produced during ripening 10 µL hydrolyzed sample injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 50 nL/min; both MALDI and ESI; Q-TOF analyzer 38 
Marine shells Proteins from layers of the shell 1 µL digested sample injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 50 nL/min; Q-TOF analyzer 39 
Saccharomyces cerevisae Proteins from whole yeast cell 2 µL digested sample injected; C18 (5 µm, 75 µm i.d. × 15 cm); ACN gradient, 350 nL/min; ion trap analyzer 40 
Human corneas Proteins from healthy and keratoconus corneas 5 µL extracted tissue injected; C18 (5 µm, 100 µm i.d. × 11 cm); ACN gradient, 500 nL/min; ion trap analyzer 41 
Preclinical mouse models Monoclonal antibodies from mouse plasma 2 µL treated sample injected; C18 (5 µm, 75 µm i.d. × 25 cm); ACN gradient, 250 nL/min; ion trap analyzer 42 
Candida albicans Surface proteins from cell wall 108 digested cells injected; C18 (3 µm, 75 µm i.d. × 15 cm); ACN gradient, 300 nL/min; TOF analyzer after UV detection 43 

*Note: Time-of-flight (TOF); quadrupole (Q); matrix-assisted laser desorption/ionization (MALDI).

Biomarkers

Biomarkers are defined as endogenous indicators of a specific biological state, usually a peptide or a carbohydrate. They can be experimentally measured and evaluated for normal or disordered processes. In the biomedical sciences, biomarkers are especially associated with healthy or diseased states. A biomarker can also be a substance introduced into an organism to estimate its normal or diseased function (44, 45).

Nano-LC plays an important role in biomarker analyses. The low analyte concentration from biological samples requires highly sensitive separation techniques and nano-LC coupled to MS or MS-MS easily presents this characteristic.

García-Villalba et al. (46) evaluated polyphenol metabolism in human breast cancer cells using nano-LC–MS. The polyphenols were found in extra virgin olive oil and their metabolites are proven to have anti-tumor activity. The authors quantified the polyphenol metabolites according to uptake time by the cancer cells and concluded that these biomarkers were easily measured by nano-LC–MS.

The search for brain trauma biomarkers in cerebrospinal fluid was proposed by Sjödin et al. (47). They measured some proteins that could indicate the level of brain trauma after a post-traumatic period by nano-LC–MS-MS. To prevent protein degradation, the autosampler was kept at 10°C. The biomarkers were enriched and quantified by the use of a commercial ligand over a wide dynamic range. However, even by using gradient elution, the chromatographic run time was too long, probably due to the high interaction between the stationary phase and the protein analytes.

From human urine, a biomarker of oxidative stress status, 8-isoprostaglandin F2α, was quantified for its indication of some diseases, such as diabetes, cancer and Alzheimer's disease (48). The authors used a microchip-based nano-HPLC, and this system involved an enrichment step before the chromatographic analysis. This enrichment promoted an increase in the MS signal, proportional to an increase in the injected concentration. The proposed method was validated and proved to be a sensitive technique for isoprostaglandin analysis.

General drugs

LC is well established as an analysis tool for pharmaceutical targets in different matrices. From drug discovery to quality assurance of drug formulations, validated LC methods have been successfully employed by the pharmaceutical industry, in research and development centers and for residual drug analysis in wastewaters (49). Although nano-LC can be used instead of typical LC, the low acceptance of this new technique at present is attributed to the high initial acquisition costs. However, the use of nano-LC is slowly increasing because of the obvious reduction in the volumes of required solvents and related waste disposal costs.

Hsieh et al. (50) determined eight common penicillin dosages in pharmaceuticals. The authors also determined these drugs in milk and tissue samples, proving the applicability of the method in different biological matrices. A packed C18 column was prepared with high repeatability for separation repetitions. Different polymeric frits were evaluated for packing the columns and polystyrene-based frits were chosen due to better separation resolution than other tested polymer frits. For the simultaneous determination of the penicillin compounds, the performance of nano-LC was compared by using both UV and MS detection (Figure 3). The limit of detection (LOD) and limit of quantification (LOQ) were higher with UV than with ion trap MS, as expected, but the repeatability of peak areas from MS was lower than with UV detection. Validation of the method using both detectors was conducted and residual penicillin drug was found in some commercial tissue samples.

Figure 3.

Chromatograms of penicillin standards or commercial products determined by nano-LC methods: penicillin signals acquired by UV detection (λ = 200 nm) (a); penicillin signals acquired by MS detection, with mass signals determined by the precursor ions (b); penicillin signals acquired by MS detection, with mass signals determined by the product ions (c); penicillin signals acquired by MS detection, with mass signals determined by the precursor ions (d). Separation conditions: sample injection of 1,000 nL, C18 silica-based column (3 µm, 100 µm i.d. × 10 cm), acetonitrile (ACN) gradient elution at a flow rate of 200 nL/min. Peaks: amoxicillin (1), ampicillin (2), penicillin G (3), penicillin (4), oxacillin (5), cloxacillin (6), nafcillin (7) and dicloxacillin (8). Reprinted from Journal of Chromatography A, Vol 1216, Author(s): Hsieh, S.-H., Huang, H.-Y., Lee, S., Determination of eight penicillin antibiotics in pharmaceuticals, milk and porcine tissues by nano-liquid chromatography, Pages 7186-7194, Copyright (2009) with permission from Elsevier.

Figure 3.

Chromatograms of penicillin standards or commercial products determined by nano-LC methods: penicillin signals acquired by UV detection (λ = 200 nm) (a); penicillin signals acquired by MS detection, with mass signals determined by the precursor ions (b); penicillin signals acquired by MS detection, with mass signals determined by the product ions (c); penicillin signals acquired by MS detection, with mass signals determined by the precursor ions (d). Separation conditions: sample injection of 1,000 nL, C18 silica-based column (3 µm, 100 µm i.d. × 10 cm), acetonitrile (ACN) gradient elution at a flow rate of 200 nL/min. Peaks: amoxicillin (1), ampicillin (2), penicillin G (3), penicillin (4), oxacillin (5), cloxacillin (6), nafcillin (7) and dicloxacillin (8). Reprinted from Journal of Chromatography A, Vol 1216, Author(s): Hsieh, S.-H., Huang, H.-Y., Lee, S., Determination of eight penicillin antibiotics in pharmaceuticals, milk and porcine tissues by nano-liquid chromatography, Pages 7186-7194, Copyright (2009) with permission from Elsevier.

D'Orazio et al. (51) performed the simultaneous determination of 18 sulfonamides, which are antimicrobial agents used for human and animal therapies. The structurally similar sulfa drugs were quantified by using nano-LC with UV and MS detection (Figure 4). The multiresidual analysis was conducted in less than 40 min at a flow rate of 190 nL/min on a C18 core-shell column, which was chosen for having better chromatographic resolution and separation efficiency than two other stationary phase options. The authors concluded that both UV and MS detection presented good detectability and the validation of the methods allowed their application in residual sulfa drugs analysis from milk samples.

Figure 4.

Comparison of nano-LC sulfonamide separations obtained while employing on-column focusing by using two detection methods: UV detection (a); MS detection (b); MS detection (c). Milk spiked with 150 µg/kg of sulfa compounds. Separation conditions: 1,000 nL of injected sample, core-shell C18 column (3 µm, 100 µm i.d. × 25 cm of packed length), ACN gradient elution at a flow rate of 190 nL/min. Peak identifications: sulfaguanidine (1), sulfanilamide (2), sulfisomidine (3), sulfadiazine (4), sulfathiazole (5), sulfapyridine (6), sulfamerazine (7), sulfamethazine (8), sulfamethizole (9), sulfameter (10), sulfamonomethoxine (11), sulfachloropyridazine (12), sulfadoxin (13), sulfamethoxazole (14), sulfisoxazole (15), sulfabenzamide (16), sulfadimethoxine (17), sulfaquinoxaline (18). Reprinted from Journal of Chromatography A, Vol 1255, Author(s): D'Orazio, G., Rocchi, S., Fanali, S. Nano-liquid chromatography coupled with mass spectrometry: separation of sulfonamides employing non-porous core-shell particles, Pages 277-285, Copyright (2012) with permission from Elsevier.

Figure 4.

Comparison of nano-LC sulfonamide separations obtained while employing on-column focusing by using two detection methods: UV detection (a); MS detection (b); MS detection (c). Milk spiked with 150 µg/kg of sulfa compounds. Separation conditions: 1,000 nL of injected sample, core-shell C18 column (3 µm, 100 µm i.d. × 25 cm of packed length), ACN gradient elution at a flow rate of 190 nL/min. Peak identifications: sulfaguanidine (1), sulfanilamide (2), sulfisomidine (3), sulfadiazine (4), sulfathiazole (5), sulfapyridine (6), sulfamerazine (7), sulfamethazine (8), sulfamethizole (9), sulfameter (10), sulfamonomethoxine (11), sulfachloropyridazine (12), sulfadoxin (13), sulfamethoxazole (14), sulfisoxazole (15), sulfabenzamide (16), sulfadimethoxine (17), sulfaquinoxaline (18). Reprinted from Journal of Chromatography A, Vol 1255, Author(s): D'Orazio, G., Rocchi, S., Fanali, S. Nano-liquid chromatography coupled with mass spectrometry: separation of sulfonamides employing non-porous core-shell particles, Pages 277-285, Copyright (2012) with permission from Elsevier.

Table II summarizes some other applications of nano-LC in current pharmaceutical analyses (52–57).

Table II

Some Applications of Nano-LC in Pharmaceutical and Other Bioactive Compound Analyses

Analyte Matrix Separation conditions and detection Reference 
Cannabinoids (psychoactive drugs) Herbal medicines 100 nL injected; C18 (4.2 µm, 100 µm i.d. × 25 cm); ACN–MeOH mixture gradient, 500 nL/min; on-column UV detection (λ = 214 nm); ESI and ion trap acquisition 52 
Antioxidants (tocopherols) Human plasma and commercial preparations 40 nL injected; C18 (3 and 5 µm; 100 µm i.d. × 56 cm); ACN–MeOH mixture gradient, flow rate not provided; on-column UV detection (λ = 205 mn) 53 
β-blockers Synthetic sample 40 nL injected; C18 (5 µm; 75 µm i.d. ×25 cm); ACN gradient, 800 nL/min; on-column UV detection (λ = 205 mn) and MS 54 
Flavonoids Human urine 100 nL injected; β-cyclodextrin based (5 µm, 100 µm i.d. × 22 cm); MeOH isocratic elution, 400 nL/min; on-column UV detection (λ = 205 nm) 55 
Non-steroidal anti-inflammatory drugs and steroids Synthetic samples and pharmaceutical formulation 100 nL injected; C18 (1.8 µm, 50–100 µm i.d. × 5 cm); ACN isocratic elution, 300–800 nL/min; on-column UV detection (λ = 200 nm) 56 
Antihypertensives Human plasma 1,000 nL injected; C18 (3 µm, 150 µm i.d. × 5 cm); ACN isocratic elution, 1,000 nL/min; MS detection (positive ion mode) 57 
Analyte Matrix Separation conditions and detection Reference 
Cannabinoids (psychoactive drugs) Herbal medicines 100 nL injected; C18 (4.2 µm, 100 µm i.d. × 25 cm); ACN–MeOH mixture gradient, 500 nL/min; on-column UV detection (λ = 214 nm); ESI and ion trap acquisition 52 
Antioxidants (tocopherols) Human plasma and commercial preparations 40 nL injected; C18 (3 and 5 µm; 100 µm i.d. × 56 cm); ACN–MeOH mixture gradient, flow rate not provided; on-column UV detection (λ = 205 mn) 53 
β-blockers Synthetic sample 40 nL injected; C18 (5 µm; 75 µm i.d. ×25 cm); ACN gradient, 800 nL/min; on-column UV detection (λ = 205 mn) and MS 54 
Flavonoids Human urine 100 nL injected; β-cyclodextrin based (5 µm, 100 µm i.d. × 22 cm); MeOH isocratic elution, 400 nL/min; on-column UV detection (λ = 205 nm) 55 
Non-steroidal anti-inflammatory drugs and steroids Synthetic samples and pharmaceutical formulation 100 nL injected; C18 (1.8 µm, 50–100 µm i.d. × 5 cm); ACN isocratic elution, 300–800 nL/min; on-column UV detection (λ = 200 nm) 56 
Antihypertensives Human plasma 1,000 nL injected; C18 (3 µm, 150 µm i.d. × 5 cm); ACN isocratic elution, 1,000 nL/min; MS detection (positive ion mode) 57 

Forensic analysis

The analyses of drugs of abuse and their metabolites in wastewaters can determine the access of the population to these substances and the public health requirements for their control. Steroid hormones, hallucinogens, cannabinoids, opioids and various prescription drugs are listed by US National Institutes on Drug Abuse as commonly used drugs of abuse (58).

Urine, sweat, blood (plasma) and saliva can be analyzed for current drug use; however, hair appears to be the best specimen, because it requires noninvasive sample collection. Compared to other specimens, a hair sample has very little possibility of adulteration and informs a longer detection period, revealing a history of drug abuse, if present (59).

Hair specimens from patients of a detoxification center were collected for the analysis of cocaine, amphetamine, morphine and related drugs (59). The authors developed a simple and validated nano-LC method as an alternative to inconclusive immunoassay techniques, using special nanochip-LC instrumentation. They also significantly reduced the sample preparation steps and the amount of sample required (less than 10% of usual quantity).

Although it is an excellent tool for monitoring, nano-LC is not usually applied for the identification and measurement of drugs of abuse, probably due to the lack of nano-LC equipment in routine analysis laboratories. Gas chromatography and conventional LC are the principal instrumental choices because of their wide distribution in forensic centers, whereas immunoassay tests are the most common analytical strategies for initial drug detection in biological samples, due to their fast and easy execution.

Enzymes

Nano-LC is still not often used for enzyme analysis (60, 61). Often, the stationary phase of nano-LC alters enzyme conformations and their catalytic activity is reduced (62). Other miniaturized techniques, such as capillary electrophoresis (CE), are preferred over nano-LC, because they do not promote alteration of the real form of the enzyme. However, reproducibility in nano-LC is higher than in CE (63), probably because the pressurized flow is more stable than the electroosmotic flow generated inside the capillary in CE.

Křížek and Kubíčková (63) reviewed the most recent methods for kinetic enzyme assays and showed that CE and its modes of separation were widely used for enzyme analysis, whereas nano-LC was used in only a few papers in recent years.

One possibility to overcome the limitations of enzyme analysis in nano-LC is the use of bioaffinity columns. These special particulate or monolithic stationary phases immobilize the enzyme in an accessible conformation without a significant loss of the original enzymatic activity (64). According to Tetala and van Beek (65), bioaffinity columns for nano-LC can easily be prepared from organic or inorganic-based materials, not only for enzymes but also for other biomolecular analyses related to the immobilized enzymes.

Conclusions and Outlook

Today, the miniaturization of analytical instrumentation presents an important role in the development of analytical sciences, which is encouraged by studies in many different areas.

Methodologies for pharmaceutical and biomedical applications must be sensitive enough to detect and quantify biologically relevant substances present in minute quantities. Especially for these low-concentration substances, the employed techniques must have excellent detectability and unquestionable identification, as provided by nano-LC–MS and nano-LC–MS-MS hyphenations.

The principal limitation at the present to wider use of nano-LC is the high cost of the analytical instrumentation. However, the rapid development of new equipment is overcoming this limitation, expanding nano-LC to routine laboratories and industries.

The chemistry of commercially available columns for nano-LC is also still a limiting factor compared to the many and versatile conventional LC columns, which cover a wide range of analytical possibilities. Stationary phase preparation, focusing on new nano columns such as monolithic and sub-2 µm particulate separation columns, is still a field that is only initiating its development.

In the near future, however, nano-LC has the potential to reach a consolidated position in the analysis of biological molecules as a complement to electrophoresis and immunoassays.

Acknowledgments

This work was supported by FAPESP, CAPES, CNPq, INCT Bioanalítica and INCTAA.

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