Editorial
For reprint orders, please contact [email protected]
Sense and nonsense of miniaturized LC–MS/MS for bioanalysis “The advantage of reducing the column diameter is not a gain in sensitivity but the ability to obtain similar detection sensitivity as with conventional LC systems with a considerable reduced sample volume.” Keywords: chromatographic elution • green chemistry • ion intensity • ion suppression • micro scale-LC • miniaturization • sensitivity enhancement
In the last two decades, conventional-sized LC coupled to tandem mass spectrometric (MS/MS) detection has become the workhorse for the quantitative determination of small-molecule drugs and metabolites in biological matrices, due to its high sensitivity and selectivity. Over that period, the typical column diameters that are routinely used have tended to decrease, from 4.6 down to 2 and even 1 mm and there is an increasing interest in reducing it further to below 1 mm (micro LC) and down to 75 μm (nano or capillary LC). Especially, the increasing interest in the field of proteomics in the last ten years has contributed to the popularity of microscale LC [1] . Several advantages are claimed to be associated with a smaller column diameter, such as reduced cost of operation, higher speed and gain in sensitivity. In order to implement the use of smaller diameter columns, other system components such as stationary phase particle size, tubing and the detector must be miniaturized as well, as demonstrated in the early papers of Knox [2] and Scott [3] . This places tighter design constrains on LC systems, mainly due to the need for reduced dead volume between connections of LC hardware. Therefore, the practical use of micro- and nanobore columns was traditionally limited to skilled technicians, who were capable of properly handling the often fragile systems that were difficult to operate in a routine setting. During the last few years, however, reliable miniaturized LC systems have become commercially available, which are able to run at low flow rates without encountering the issues mentioned
10.4155/BIO.14.263 © 2014 Future Science Ltd
above. It is therefore a good time to re-evaluate the potential of micro- and nano-LC for the field of quantitative bioanalysis of small molecules. Sensitivity & sample volume An often reported benefit of reducing column diameter, is the gain in signal-to-noise when concentration-sensitive detectors are employed such as ESI–MS/MS [4,5] . The reason behind this is that the chromatographic dilution of the analyte is significantly reduced as the analytes elute in a smaller volume. The equation describing the chromatographic dilution is shown below [6] : C end = D = C inj
2
frr (1 + K ) V inj
Martijn Hilhorst Author for correspondence: PRA Health Sciences, Westerbrink, The Netherlands Tel.: + 31 592303441 Fax: + 31 303222 hilhorstmartijn@ PRAHS.com
Chad Briscoe PRA Health Sciences, Lenexa, KS, USA
2rLH Equation 1
where ε is the column porosity, r the column radius, k the retention factor, L the column length, H the plate height and Vinj the injection volume. Thus, by reducing the column diameter, the dilution of an analyte eluting from the column decreases proportionally to r2 and, as a consequence, the analyte concentration in the eluting peak volume increases with the same factor. For example, reducing the column diameter from 4.6 mm to 1.0 mm results in a 21-fold increase of the peak concentration, when the same volume of sample is injected. Unfortunately, the injectable volume (Vinj in the equation) will also decrease proportionally to r2 due to the reduced loadability of the column at smaller radius and the injection volume needs to be decreased as reported by Vissers [7] . As column
Bioanalysis (2014) 6(24), 3263–3265
Nico van de Merbel PRA Health Sciences, Westerbrink, The Netherlands Department of Analytical and Biochemistry, Centre for Pharmacy, University of Groningen, Groningen, The Netherlands
part of
ISSN 1757-6180
3263
Editorial Hilhorst, Briscoe & van de Merbel loadability decreases to the same extent, no net sensitivity gain will be obtained by miniaturizing the column diameter. It is often argued that the overloading can be compensated by applying preconcentration techniques such as on-line SPE or on-column focusing. However, if the properties of compound, solvent, mobile phase and type of column allow focusing, the same strategy can also be applied using a conventional sized LC system, provided that enough sample is present.
“
Although the advantages of microscale chromatography are clear, the robustness of the used equipment needs to be further demonstrated before it can be expected to be widely introduced in a routine bioanalytical setting.
”
It is therefore fair to say that the advantage of reducing the diameter is not a gain in sensitivity, but the ability to obtain similar detection sensitivity as with a conventional LC system with a considerably reduced sample volume. As a consequence, to truly evaluate the value of micro- and nanobore LC it is important to assess the need to analyze small volumes in a routine bioanalytical lab. For many clinical trials, the amount of plasma available for analysis is typically more than a milliliter. Using common sample pretreatment procedures, a sample extract volume of at least 100 μl is usually obtained and this allows multiple injections on a standard LC column to obtain the sensitivity needed for the majority of our applications. Therefore, downscaling column diameter does not have any sensitivity benefits as there is ample injection volume available. Nevertheless, the ability to analyze very small samples is of great importance to other fields of bioanalysis, where the volume of samples is limited such as in studies with small animals or children and/or with matrices for which only small samples can be collected [8] . Especially when it is desirable to perform reanalysis of a small sample, switching to micro- or nano-LC may be the only way to go. Operational aspects MS source
In quantitative bioanalysis with LC–MS, ESI is often used as the ionization technique. One of the features of ESI is that it makes the MS behave like a concentrationsensitive detector. Therefore, the signal is not influenced by the flow rate, making it an ideal detector for miniaturized systems. Miniaturized chromatography also eliminates the need for splitting the mobile phase, as is occasionally needed in conventional LC–MS to limit the amount of volume introduced into the mass spectrometer, especially for the older types of instruments. When splitting the flow, the ratio of the volumes
3264
Bioanalysis (2014) 6(24)
sent to the MS and to waste depends on the length and diameter of the tubing used, the actual amount entering the MS may vary from batch to batch. Also, small plugs in the tubing can cause significant changes in the split ratio mid-run. Using miniaturized LC, splitting of the mobile phase becomes unnecessary, thereby simplifying introduction into the MS. Another advantage of micro- and nano-LC, is the MS interface does not get as dirty as with conventional LC. It is commonly known that ion sources tend to contaminate over time due to mobile phase salts and bioanalytical samples that contain traces of biological matrix. This leads to the need for cleaning and down-time of the LC–MS system. At the lower flow rates used with smaller column diameters, smaller injection volumes and less solvent is introduced in the MS source, which keeps the ion source cleaner for a longer period of time. Like LC systems, commercial MS ion sources are available that are optimized for the use with low flow rates. Several authors have reported advantages in the Electrospray Ionization (ESI) source characteristics at reduced low flow-rates as applied to microscale chromatography [9–13] . It was discovered that the lower flow rate reduced the size of the charged droplets produced in the spraying process, which results in an improved evaporation and fission cascade and ultimately to higher ionization efficiencies, less ion suppression and more tolerance towards the presents of salt in the mobile phase [11] . For example, Heemskerk et al. [13] showed an increase in ion intensity of about five-fold for a model phosphopeptide when flow rate dropped ten-fold and Gangl et al. [10] demonstrated a suppression decrease from 60 to 30% in the analysis of Carveditol in the presence of a suppressing agent after reducing the flow rate from 200 to 0.1 μl/min. These effects can only be fully utilized when using ESI sources specifically designed for low flow rates. Robustness
Several practical issues need to be considered when implementing micro-scale LC–MS in a routine bioanalytical setting. Due to the small volumes and flow rates, the effect of dead volumes is much more pronounced than in conventional LC–MS. This results in the need for high-quality fluidic connections, which usually are difficult – if not impossible – to prepare by the users themselves. Also, the chance of clogging somewhere in the flow path increases by reducing tubing diameters. If connections fail and leakage occurs, this might not be visible by the eye because of the very low flow rates, which making trouble shooting difficult. In this respect, it is valuable that ready-to-use commercial instrumentation is available from several vendors to (at least partly) solve these issues and help
future science group
Sense & nonsense of miniaturized LC–MS/MS for bioanalysis
improve the robustness of miniaturized LC–MS in a routine bioanalytical laboratory. Reduction of cost/green chemistry The use of low-volume chromatography has attracted interest in the pharmaceutical industry due to its economic and environmental advantages, a development commonly known as ‘green chemistry’ [14] . As the column diameter decreases, so does the volume of mobile phase, which can lead to a considerable decrease in operational costs, especially when expensive mobile phase components are used such ultrapure modifiers or chiral additives. Likewise, reduced volumes of mobile phase obviously also are less of an environmental burden. As an example, a conventional 4.6 mm column typically operates at a flow-rate of 1 ml/min and will require about 1.5 l of solvent a day, a large percentage of which is an organic modifier [14] . A micro-LC system using a 75 μm column only requires 25 ml of solvent per day. Depending on the percentage and type of modifier, this can represent a large cost saving on a yearly basis. However, it must be noted that this reduction does not apply when conventional size pumps equipped with a flow splitter are used. Conclusion From a scientific point of view, the usefulness of reducing LC column diameter for routine bioanalysis largely depends on the sample (injection) volume available. If
there is sufficient sample, no significant gain in sensitivity will be obtained by the use of micro- or nano-bore LC–MS. On the other hand, when the sample volume is limited, injection onto a conventional column may lead to undesirable chromatographic dilution and miniaturization can be an attractive alternative that should be explored in case sensitivity needs to be improved. From a practical point of view, miniaturization will lead to less waste to dispose and to a reduction of solvent costs. Even though commercially available microLC systems have improved considerably over the last decade in terms of robustness, the importance of eliminating dead volumes and the associated risks in case of failure are still high compared with conventional LC systems. Although the advantages of microscale chromatography are clear, the robustness of this equipment needs to be further demonstrated before it can be expected to be widely introduced in a routine bioanalytical setting. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript. In: Handbook of LC–MS Bioanalysis; Best Practices Experimental Protocols and Regulations. John Wiley and Sons Inc., NY, USA, 307–316 (2013).
References 1
Rainville P. Microfluid LC–MS for analysis of small-volume biofluid samples: where we have been and where we need to go. Bioanalysis 3(1), 1–3, (2011).
2
Knox JH. Theoritical aspects of LC with packed and open small-bore columns. J. Chromatogr. Sci. 18, 453–461 (1980).
3
Reese CE, Scott RPW. Microbore Columns – design, construction, and operation. J. Chromatogr. Sci. 18, 479–486 (1980).
4
Arnold DW, Needham SR. Micro-LC–MS-MS: the future of bioanalysis. Bioanalysis 5(11), 1329–1331 (2013).
5
Christianson CC, Johnson CJL, Needham SR. The advantages of microflow LC–MS/MS compared with conventional HPLC–MS/MS for the analysis of methotrexate from human plasma. Bioanalysis 5(11), 1387–1396, (2013).
6
Qu J, Qu Y, Straubinger RM. Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid phase extraction and reversed-phase capilary high performance liquid chromatography/tandem mass spectrometry. Anal. Chem. 79, 3786–3793 (2007).
7
8
Vissers PC, Cleassens HA, Cramers CA. Microcolumn liquid chromatography: instrumentation, detection and applications. J. Chromatogr. A 779, 1–28 (1997). Skor H, Rahavendran SV. Microflow LC–MS for quantitative analysis of drugs in support of microsampling,
future science group
Editoral
9
Valaskovic GA, Utley L, Lee MS, Wu JT. Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimantion of metabolite levels in drug discovery. Rapid Commun. Mass Spectrom. 20, 1087–1096 (2006).
10
Gangl ET, Anan M, Spooner Neil, Vouros P. Reduction of signal suppression effects in ESI-MS using a nanosplitting device. Anal. Chem. 73, 5635–5644 (2001).
11
Juraschek R, Dülcks T, Karas M. Nanoelectrospray – more than just a minimized-flow electrospray ionization source. J. Am. Soc. Mass Spectrom. 10, 300–3008 (1999).
12
Schmidt A, Karas M. Effect of different solution flow rates on analyte ion signals in nano-ESI-MS, or: when does ESI turn into nano-ESI? J. Am. Soc. Mass Spectrom. 14, 492–500 (2003).
13
Heemskerk AAM, Busnel JM, Schoenmaker B et al. Ultralow flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal. Chem. 84, 4552–4559 (2012).
14
Ghosh C. Green bioanalysis: some innovative ideas towards green analytical techniques. Bioanalysis 4(11), 1377–1391 (2012).
www.future-science.com
3265
For reprint orders, please contact [email protected]
Sense and nonsense of miniaturized LC–MS/MS for bioanalysis “The advantage of reducing the column diameter is not a gain in sensitivity but the ability to obtain similar detection sensitivity as with conventional LC systems with a considerable reduced sample volume.” Keywords: chromatographic elution • green chemistry • ion intensity • ion suppression • micro scale-LC • miniaturization • sensitivity enhancement
In the last two decades, conventional-sized LC coupled to tandem mass spectrometric (MS/MS) detection has become the workhorse for the quantitative determination of small-molecule drugs and metabolites in biological matrices, due to its high sensitivity and selectivity. Over that period, the typical column diameters that are routinely used have tended to decrease, from 4.6 down to 2 and even 1 mm and there is an increasing interest in reducing it further to below 1 mm (micro LC) and down to 75 μm (nano or capillary LC). Especially, the increasing interest in the field of proteomics in the last ten years has contributed to the popularity of microscale LC [1] . Several advantages are claimed to be associated with a smaller column diameter, such as reduced cost of operation, higher speed and gain in sensitivity. In order to implement the use of smaller diameter columns, other system components such as stationary phase particle size, tubing and the detector must be miniaturized as well, as demonstrated in the early papers of Knox [2] and Scott [3] . This places tighter design constrains on LC systems, mainly due to the need for reduced dead volume between connections of LC hardware. Therefore, the practical use of micro- and nanobore columns was traditionally limited to skilled technicians, who were capable of properly handling the often fragile systems that were difficult to operate in a routine setting. During the last few years, however, reliable miniaturized LC systems have become commercially available, which are able to run at low flow rates without encountering the issues mentioned
10.4155/BIO.14.263 © 2014 Future Science Ltd
above. It is therefore a good time to re-evaluate the potential of micro- and nano-LC for the field of quantitative bioanalysis of small molecules. Sensitivity & sample volume An often reported benefit of reducing column diameter, is the gain in signal-to-noise when concentration-sensitive detectors are employed such as ESI–MS/MS [4,5] . The reason behind this is that the chromatographic dilution of the analyte is significantly reduced as the analytes elute in a smaller volume. The equation describing the chromatographic dilution is shown below [6] : C end = D = C inj
2
frr (1 + K ) V inj
Martijn Hilhorst Author for correspondence: PRA Health Sciences, Westerbrink, The Netherlands Tel.: + 31 592303441 Fax: + 31 303222 hilhorstmartijn@ PRAHS.com
Chad Briscoe PRA Health Sciences, Lenexa, KS, USA
2rLH Equation 1
where ε is the column porosity, r the column radius, k the retention factor, L the column length, H the plate height and Vinj the injection volume. Thus, by reducing the column diameter, the dilution of an analyte eluting from the column decreases proportionally to r2 and, as a consequence, the analyte concentration in the eluting peak volume increases with the same factor. For example, reducing the column diameter from 4.6 mm to 1.0 mm results in a 21-fold increase of the peak concentration, when the same volume of sample is injected. Unfortunately, the injectable volume (Vinj in the equation) will also decrease proportionally to r2 due to the reduced loadability of the column at smaller radius and the injection volume needs to be decreased as reported by Vissers [7] . As column
Bioanalysis (2014) 6(24), 3263–3265
Nico van de Merbel PRA Health Sciences, Westerbrink, The Netherlands Department of Analytical and Biochemistry, Centre for Pharmacy, University of Groningen, Groningen, The Netherlands
part of
ISSN 1757-6180
3263
Editorial Hilhorst, Briscoe & van de Merbel loadability decreases to the same extent, no net sensitivity gain will be obtained by miniaturizing the column diameter. It is often argued that the overloading can be compensated by applying preconcentration techniques such as on-line SPE or on-column focusing. However, if the properties of compound, solvent, mobile phase and type of column allow focusing, the same strategy can also be applied using a conventional sized LC system, provided that enough sample is present.
“
Although the advantages of microscale chromatography are clear, the robustness of the used equipment needs to be further demonstrated before it can be expected to be widely introduced in a routine bioanalytical setting.
”
It is therefore fair to say that the advantage of reducing the diameter is not a gain in sensitivity, but the ability to obtain similar detection sensitivity as with a conventional LC system with a considerably reduced sample volume. As a consequence, to truly evaluate the value of micro- and nanobore LC it is important to assess the need to analyze small volumes in a routine bioanalytical lab. For many clinical trials, the amount of plasma available for analysis is typically more than a milliliter. Using common sample pretreatment procedures, a sample extract volume of at least 100 μl is usually obtained and this allows multiple injections on a standard LC column to obtain the sensitivity needed for the majority of our applications. Therefore, downscaling column diameter does not have any sensitivity benefits as there is ample injection volume available. Nevertheless, the ability to analyze very small samples is of great importance to other fields of bioanalysis, where the volume of samples is limited such as in studies with small animals or children and/or with matrices for which only small samples can be collected [8] . Especially when it is desirable to perform reanalysis of a small sample, switching to micro- or nano-LC may be the only way to go. Operational aspects MS source
In quantitative bioanalysis with LC–MS, ESI is often used as the ionization technique. One of the features of ESI is that it makes the MS behave like a concentrationsensitive detector. Therefore, the signal is not influenced by the flow rate, making it an ideal detector for miniaturized systems. Miniaturized chromatography also eliminates the need for splitting the mobile phase, as is occasionally needed in conventional LC–MS to limit the amount of volume introduced into the mass spectrometer, especially for the older types of instruments. When splitting the flow, the ratio of the volumes
3264
Bioanalysis (2014) 6(24)
sent to the MS and to waste depends on the length and diameter of the tubing used, the actual amount entering the MS may vary from batch to batch. Also, small plugs in the tubing can cause significant changes in the split ratio mid-run. Using miniaturized LC, splitting of the mobile phase becomes unnecessary, thereby simplifying introduction into the MS. Another advantage of micro- and nano-LC, is the MS interface does not get as dirty as with conventional LC. It is commonly known that ion sources tend to contaminate over time due to mobile phase salts and bioanalytical samples that contain traces of biological matrix. This leads to the need for cleaning and down-time of the LC–MS system. At the lower flow rates used with smaller column diameters, smaller injection volumes and less solvent is introduced in the MS source, which keeps the ion source cleaner for a longer period of time. Like LC systems, commercial MS ion sources are available that are optimized for the use with low flow rates. Several authors have reported advantages in the Electrospray Ionization (ESI) source characteristics at reduced low flow-rates as applied to microscale chromatography [9–13] . It was discovered that the lower flow rate reduced the size of the charged droplets produced in the spraying process, which results in an improved evaporation and fission cascade and ultimately to higher ionization efficiencies, less ion suppression and more tolerance towards the presents of salt in the mobile phase [11] . For example, Heemskerk et al. [13] showed an increase in ion intensity of about five-fold for a model phosphopeptide when flow rate dropped ten-fold and Gangl et al. [10] demonstrated a suppression decrease from 60 to 30% in the analysis of Carveditol in the presence of a suppressing agent after reducing the flow rate from 200 to 0.1 μl/min. These effects can only be fully utilized when using ESI sources specifically designed for low flow rates. Robustness
Several practical issues need to be considered when implementing micro-scale LC–MS in a routine bioanalytical setting. Due to the small volumes and flow rates, the effect of dead volumes is much more pronounced than in conventional LC–MS. This results in the need for high-quality fluidic connections, which usually are difficult – if not impossible – to prepare by the users themselves. Also, the chance of clogging somewhere in the flow path increases by reducing tubing diameters. If connections fail and leakage occurs, this might not be visible by the eye because of the very low flow rates, which making trouble shooting difficult. In this respect, it is valuable that ready-to-use commercial instrumentation is available from several vendors to (at least partly) solve these issues and help
future science group
Sense & nonsense of miniaturized LC–MS/MS for bioanalysis
improve the robustness of miniaturized LC–MS in a routine bioanalytical laboratory. Reduction of cost/green chemistry The use of low-volume chromatography has attracted interest in the pharmaceutical industry due to its economic and environmental advantages, a development commonly known as ‘green chemistry’ [14] . As the column diameter decreases, so does the volume of mobile phase, which can lead to a considerable decrease in operational costs, especially when expensive mobile phase components are used such ultrapure modifiers or chiral additives. Likewise, reduced volumes of mobile phase obviously also are less of an environmental burden. As an example, a conventional 4.6 mm column typically operates at a flow-rate of 1 ml/min and will require about 1.5 l of solvent a day, a large percentage of which is an organic modifier [14] . A micro-LC system using a 75 μm column only requires 25 ml of solvent per day. Depending on the percentage and type of modifier, this can represent a large cost saving on a yearly basis. However, it must be noted that this reduction does not apply when conventional size pumps equipped with a flow splitter are used. Conclusion From a scientific point of view, the usefulness of reducing LC column diameter for routine bioanalysis largely depends on the sample (injection) volume available. If
there is sufficient sample, no significant gain in sensitivity will be obtained by the use of micro- or nano-bore LC–MS. On the other hand, when the sample volume is limited, injection onto a conventional column may lead to undesirable chromatographic dilution and miniaturization can be an attractive alternative that should be explored in case sensitivity needs to be improved. From a practical point of view, miniaturization will lead to less waste to dispose and to a reduction of solvent costs. Even though commercially available microLC systems have improved considerably over the last decade in terms of robustness, the importance of eliminating dead volumes and the associated risks in case of failure are still high compared with conventional LC systems. Although the advantages of microscale chromatography are clear, the robustness of this equipment needs to be further demonstrated before it can be expected to be widely introduced in a routine bioanalytical setting. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript. In: Handbook of LC–MS Bioanalysis; Best Practices Experimental Protocols and Regulations. John Wiley and Sons Inc., NY, USA, 307–316 (2013).
References 1
Rainville P. Microfluid LC–MS for analysis of small-volume biofluid samples: where we have been and where we need to go. Bioanalysis 3(1), 1–3, (2011).
2
Knox JH. Theoritical aspects of LC with packed and open small-bore columns. J. Chromatogr. Sci. 18, 453–461 (1980).
3
Reese CE, Scott RPW. Microbore Columns – design, construction, and operation. J. Chromatogr. Sci. 18, 479–486 (1980).
4
Arnold DW, Needham SR. Micro-LC–MS-MS: the future of bioanalysis. Bioanalysis 5(11), 1329–1331 (2013).
5
Christianson CC, Johnson CJL, Needham SR. The advantages of microflow LC–MS/MS compared with conventional HPLC–MS/MS for the analysis of methotrexate from human plasma. Bioanalysis 5(11), 1387–1396, (2013).
6
Qu J, Qu Y, Straubinger RM. Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid phase extraction and reversed-phase capilary high performance liquid chromatography/tandem mass spectrometry. Anal. Chem. 79, 3786–3793 (2007).
7
8
Vissers PC, Cleassens HA, Cramers CA. Microcolumn liquid chromatography: instrumentation, detection and applications. J. Chromatogr. A 779, 1–28 (1997). Skor H, Rahavendran SV. Microflow LC–MS for quantitative analysis of drugs in support of microsampling,
future science group
Editoral
9
Valaskovic GA, Utley L, Lee MS, Wu JT. Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimantion of metabolite levels in drug discovery. Rapid Commun. Mass Spectrom. 20, 1087–1096 (2006).
10
Gangl ET, Anan M, Spooner Neil, Vouros P. Reduction of signal suppression effects in ESI-MS using a nanosplitting device. Anal. Chem. 73, 5635–5644 (2001).
11
Juraschek R, Dülcks T, Karas M. Nanoelectrospray – more than just a minimized-flow electrospray ionization source. J. Am. Soc. Mass Spectrom. 10, 300–3008 (1999).
12
Schmidt A, Karas M. Effect of different solution flow rates on analyte ion signals in nano-ESI-MS, or: when does ESI turn into nano-ESI? J. Am. Soc. Mass Spectrom. 14, 492–500 (2003).
13
Heemskerk AAM, Busnel JM, Schoenmaker B et al. Ultralow flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics. Anal. Chem. 84, 4552–4559 (2012).
14
Ghosh C. Green bioanalysis: some innovative ideas towards green analytical techniques. Bioanalysis 4(11), 1377–1391 (2012).
www.future-science.com
3265
