- Email: [email protected]
Micro scale analytical derivatizations on solid phase
Accepted Manuscript Micro Scale Analytical Derivatizations on Solid Phase Sanka N. Atapattu, Jack M. Rosenfeld PII:
S0165-9936(18)30512-0
DOI:
https://doi.org/10.1016/j.trac.2018.10.028
Reference:
TRAC 15292
To appear in:
Trends in Analytical Chemistry
Received Date: 27 September 2018 Revised Date:
24 October 2018
Accepted Date: 25 October 2018
Please cite this article as: S.N. Atapattu, J.M. Rosenfeld, Micro Scale Analytical Derivatizations on Solid Phase, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2018.10.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Micro Scale Analytical Derivatizations on Solid Phase
2
34 35 36 37 38
By Sanka N. Atapattu* and 2Jack M. Rosenfeld
1
Department of Pathology and Molecular Medicine McMaster University Hamilton, ON L8S 4K1 Canada
TE D
M AN U
2
SC
CanAm Bioresearch Inc. Winnipeg, MB R3T 0P4 Canada
RI PT
1
EP
Address for correspondence: Sanka N. Atapattu CanAm BioResearch Inc. 6-1200 Waverley Street Winnipeg, MB R3T 0P4 Tel: 204-599-5434 Fax: 204-488-9823 E-mail: [email protected]
AC C
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
ACCEPTED MANUSCRIPT 2 Abstract
40
Analytical derivatization (AD), a subset of functional group analysis, alters the structure of an
41
analyte to a product more suitable for analysis. The reactions impart stability to the product and a
42
functionality that enhances sensitivity and specificity to the determinations. By targeting the
43
functional groups for labeling AD adds selectivity to the measurements. Associated with these
44
advantages, however, is the additional step required in sample preparation. The issue of the extra
45
step was resolved by solid phase analytical derivatization (SPAD) which combined the extraction
46
and derivatization step or limited the process to a one-pot reaction in which extraction and
47
derivatization occurred on the solid phase without intermediate isolation of the analyte. Within
48
the broad class of organic acids, SPAD provides conditions that can: derivatize both
49
functionalities; selectively derivatize carboxylic acids in the presence of phenols by reaction at
50
neutral pH which does not ionize phenols; derivatize phenols in the presence of acids by
51
selectively inhibiting derivatization of carboxylic acids at alkaline pH. These reaction
52
characteristics hold whether carboxylic acids are on different compounds or on the same
53
compounds. In the case of carbonyls, SPAD enhances the reaction rate over solution chemistry
54
so that both aldehydes and the usually slower reacting ketones are rapidly extracted/derivatized
55
in one step. The derivatization incorporates chromophores, fluorophores and electrophores for
56
purpose of detection, as well lipophilicity for purpose of extraction. In the case of primary and
57
secondary amines, SPAD functional groups that enhance sensitivity and/or lipophilicity. Initially,
58
SPAD was a batch process but transitioned into a microextraction/derivatization and an
59
automated technique.
64 65
SC
M AN U
TE D
EP
61 62 63
Key words: Analytical derivatization, Solid phase analytical derivatization, Sample preparation, Microextraction methods.
AC C
60
RI PT
39
1. Introduction
Solid phase extractions (SPE) were developed to overcome the well-known difficulties of
66
liquid/liquid extractions (LLE). It is a successful and valuable technique with wide applications
67
in bio-analytical and environmental problems. There are, however, classes of analytes that
68
cannot be effectively determined by sorption/desorption alone but require derivatization. Solid
69
phase analytical derivatizations (SPAD) add an extra dimension to SPE while maintaining its
70
advantages [1-3]. It is a green chemistry with low or no organic solvent consumption and SPAD
ACCEPTED MANUSCRIPT 3 71
reduces cost by raising efficiency.
72
regularly reviewed [1-3].
These are significant advantages.
The field has been
The reagents used in SPAD impart increased sensitivity of detection and/or stability to
74
the derivatives. In chromatographic analysis when analyte molecules have unfavorable
75
interactions with the LC or GC stationary phase can result in loss of sensitivity, poor resolution
76
and peak tailing [1]. Derivatizing the target analyte with a suitable reagent would not only
77
improve peak resolution, peak shape but also achieve better sensitivity. This is an important
78
aspect in trace level analysis when analysts have restrictions with instrument sensitivity [1,3].
RI PT
73
In the case of hydrophilic compounds, the derivatives are more lipophilic which
80
facilitates isolation. The reactions occur at ambient temperature and in the case of ketones, are
81
often considerably faster than those in solution. These mild conditions are particularly important
82
for derivatization of potentially unstable compounds such as prostaglandin E2. Because reagents
83
are added to the reaction mixture there is great flexibility in matching reagents to analyte.
M AN U
SC
79
An important variant of SPAD facilitated glycomic investigations [4,5]. Glycoproteins
85
and glycopeptides were bound onto Aminolink 4% a cross linked beaded agarose bead with
86
aldehyde functionality. The terminal amino groups on macromolecule react with the aldehyde to
87
form an imine which is reduced to an amine by NaCNBH4 to immobilize the protein or peptide.
88
Despite the immobililzation, the glycoproteins and glyopeptides were reactive to both
89
derivatizing reagents and – perhaps surprisingly to hydrolytic enzymes in solutions added to the
90
resin. The reactivity allowed modifications of sialic acids which included, esterification with
91
ethanol and subsequent amidation with p-toluidine both under catalysis by N-(3-
92
(dimethylamino)propyl)-N′-ethylcarbodiimidehydrochloride (EDC). These derivatizations were
93
instrumental in differentiating between α2,3 and α2,6 linkages of sialic acids which are
94
recognition molecules for viral infections. The N-glycans were enzymatically cleaved and eluted
95
from the beads using the well-known PNGase procedure. The O- glycans were cleaved and
96
eluted from the beads by a newly discovered enzyme termed Operator [4,5]. This demonstrate
97
that the enzymatic activity on the solid phase was not limited to PNGase.
AC C
EP
TE D
84
98
Solid phase configurations explored during development and use of SPAD included
99
beads suspended in a liquid, beads packed into columns, solid phases on filters or as a thin film
100
on a fiber for solid phase microextraction (SPME) [1-3].
Initial SPAD techniques were batch
ACCEPTED MANUSCRIPT 4 101
processes but these transitioned to a column configuration and full automation as a miniaturized
102
technique. Despite the advantages of SPAD, there remains the critique that large excess of reagents
104
are required. While these are often chromatographically well separated from the derivatives, the
105
excess reagent can contribute contaminants with potential for interference to analysis. Excess of
106
reagent may be required to achieve complete reaction and require cleanup protocols. The
107
reagent excess can compromise the reusability of the solid phase – for instance shortening the
108
life time of the SPME fiber. Use of reagents also precludes direct in-vivo sampling. However,
109
extraction followed by derivatization of the sorbed analyte can provide advantages of SPAD
110
without exposing the organism to reactive compounds.
112 113
SC
This review covers microextraction applications of SPAD with a focus on literature
M AN U
111
RI PT
103
published 2017-2018.
2. Solid phase analytical derivatization early developments
The XAD resins formulated as beads were widely used in SPE applications due to their
115
high surface area and high absorptivity of lipophilic analytes and reagents mainly through
116
hydrophobic bonding and concentrating a smaller volume of beads. In addition, the XAD resins
117
are stable to acidic and alkaline. The first SPAD reaction reported in 1984 as well as subsequent
118
methods used XAD-2, a styrene–divinylbenzene crosslinked polymeric macroreticular resin as
119
the solid phase [6]. Use of XAD resins with different physical or chemical characteristics did
120
not improve yield or reaction rate.
TE D
114
A variety of reagents and analytes were tested. Pentafluorobenzyl bromide (PFBBr)
122
served to form PFB esters of carboxylic acids or PFB ethers of phenols such as
123
tetrahydrocannabinol and other cannabinoids [1-3]. Derivatization at alkaline pH using PFBBr
124
produced PFB esters of carboxylic acids and PFB ethers of phenols. At neutral pH the SPAD
125
yielded only the esters of carboxylic acids. Addition of small volumes of C1-C5 alcohols to a
126
SPAD reaction at alkaline pH suppressed the formation of the esters but not of phenols thus
127
providing enhanced specificity to the analysis. Derivatization with pentafluoropyridine (PFP)
128
under alkaline SPAD conditions was selective towards phenols in the presence of carboxylic
129
acids. Pentafluorobenzyl hydroxyl amine (PFBONH2), benzyl hydroxylamine (BzONH2) rapidly
130
form PFBoxime or Bzoximes of aldehydes and ketones ranging in size from C1-C20.
AC C
EP
121
Under
ACCEPTED MANUSCRIPT 5 131
acidic SPAD 2,4 dinitrophenylhydrazine (DNPH) rapidly formed the hydrazone product of both
132
aldehydes and ketones. In recent years researchers have explored novel solid phases for SPAD. These included
134
solid phase microextraction fibers, various nanoparticles, monolith columns, metals, metal
135
oxides, glass plates, GC injection ports, hollow fibers, stir bars and ionic liquids. Some of the
136
key milestones in SPAD method development are listed in Table 01.
137
S0165-9936(18)30512-0
DOI:
https://doi.org/10.1016/j.trac.2018.10.028
Reference:
TRAC 15292
To appear in:
Trends in Analytical Chemistry
Received Date: 27 September 2018 Revised Date:
24 October 2018
Accepted Date: 25 October 2018
Please cite this article as: S.N. Atapattu, J.M. Rosenfeld, Micro Scale Analytical Derivatizations on Solid Phase, Trends in Analytical Chemistry, https://doi.org/10.1016/j.trac.2018.10.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Micro Scale Analytical Derivatizations on Solid Phase
2
34 35 36 37 38
By Sanka N. Atapattu* and 2Jack M. Rosenfeld
1
Department of Pathology and Molecular Medicine McMaster University Hamilton, ON L8S 4K1 Canada
TE D
M AN U
2
SC
CanAm Bioresearch Inc. Winnipeg, MB R3T 0P4 Canada
RI PT
1
EP
Address for correspondence: Sanka N. Atapattu CanAm BioResearch Inc. 6-1200 Waverley Street Winnipeg, MB R3T 0P4 Tel: 204-599-5434 Fax: 204-488-9823 E-mail: [email protected]
AC C
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
ACCEPTED MANUSCRIPT 2 Abstract
40
Analytical derivatization (AD), a subset of functional group analysis, alters the structure of an
41
analyte to a product more suitable for analysis. The reactions impart stability to the product and a
42
functionality that enhances sensitivity and specificity to the determinations. By targeting the
43
functional groups for labeling AD adds selectivity to the measurements. Associated with these
44
advantages, however, is the additional step required in sample preparation. The issue of the extra
45
step was resolved by solid phase analytical derivatization (SPAD) which combined the extraction
46
and derivatization step or limited the process to a one-pot reaction in which extraction and
47
derivatization occurred on the solid phase without intermediate isolation of the analyte. Within
48
the broad class of organic acids, SPAD provides conditions that can: derivatize both
49
functionalities; selectively derivatize carboxylic acids in the presence of phenols by reaction at
50
neutral pH which does not ionize phenols; derivatize phenols in the presence of acids by
51
selectively inhibiting derivatization of carboxylic acids at alkaline pH. These reaction
52
characteristics hold whether carboxylic acids are on different compounds or on the same
53
compounds. In the case of carbonyls, SPAD enhances the reaction rate over solution chemistry
54
so that both aldehydes and the usually slower reacting ketones are rapidly extracted/derivatized
55
in one step. The derivatization incorporates chromophores, fluorophores and electrophores for
56
purpose of detection, as well lipophilicity for purpose of extraction. In the case of primary and
57
secondary amines, SPAD functional groups that enhance sensitivity and/or lipophilicity. Initially,
58
SPAD was a batch process but transitioned into a microextraction/derivatization and an
59
automated technique.
64 65
SC
M AN U
TE D
EP
61 62 63
Key words: Analytical derivatization, Solid phase analytical derivatization, Sample preparation, Microextraction methods.
AC C
60
RI PT
39
1. Introduction
Solid phase extractions (SPE) were developed to overcome the well-known difficulties of
66
liquid/liquid extractions (LLE). It is a successful and valuable technique with wide applications
67
in bio-analytical and environmental problems. There are, however, classes of analytes that
68
cannot be effectively determined by sorption/desorption alone but require derivatization. Solid
69
phase analytical derivatizations (SPAD) add an extra dimension to SPE while maintaining its
70
advantages [1-3]. It is a green chemistry with low or no organic solvent consumption and SPAD
ACCEPTED MANUSCRIPT 3 71
reduces cost by raising efficiency.
72
regularly reviewed [1-3].
These are significant advantages.
The field has been
The reagents used in SPAD impart increased sensitivity of detection and/or stability to
74
the derivatives. In chromatographic analysis when analyte molecules have unfavorable
75
interactions with the LC or GC stationary phase can result in loss of sensitivity, poor resolution
76
and peak tailing [1]. Derivatizing the target analyte with a suitable reagent would not only
77
improve peak resolution, peak shape but also achieve better sensitivity. This is an important
78
aspect in trace level analysis when analysts have restrictions with instrument sensitivity [1,3].
RI PT
73
In the case of hydrophilic compounds, the derivatives are more lipophilic which
80
facilitates isolation. The reactions occur at ambient temperature and in the case of ketones, are
81
often considerably faster than those in solution. These mild conditions are particularly important
82
for derivatization of potentially unstable compounds such as prostaglandin E2. Because reagents
83
are added to the reaction mixture there is great flexibility in matching reagents to analyte.
M AN U
SC
79
An important variant of SPAD facilitated glycomic investigations [4,5]. Glycoproteins
85
and glycopeptides were bound onto Aminolink 4% a cross linked beaded agarose bead with
86
aldehyde functionality. The terminal amino groups on macromolecule react with the aldehyde to
87
form an imine which is reduced to an amine by NaCNBH4 to immobilize the protein or peptide.
88
Despite the immobililzation, the glycoproteins and glyopeptides were reactive to both
89
derivatizing reagents and – perhaps surprisingly to hydrolytic enzymes in solutions added to the
90
resin. The reactivity allowed modifications of sialic acids which included, esterification with
91
ethanol and subsequent amidation with p-toluidine both under catalysis by N-(3-
92
(dimethylamino)propyl)-N′-ethylcarbodiimidehydrochloride (EDC). These derivatizations were
93
instrumental in differentiating between α2,3 and α2,6 linkages of sialic acids which are
94
recognition molecules for viral infections. The N-glycans were enzymatically cleaved and eluted
95
from the beads using the well-known PNGase procedure. The O- glycans were cleaved and
96
eluted from the beads by a newly discovered enzyme termed Operator [4,5]. This demonstrate
97
that the enzymatic activity on the solid phase was not limited to PNGase.
AC C
EP
TE D
84
98
Solid phase configurations explored during development and use of SPAD included
99
beads suspended in a liquid, beads packed into columns, solid phases on filters or as a thin film
100
on a fiber for solid phase microextraction (SPME) [1-3].
Initial SPAD techniques were batch
ACCEPTED MANUSCRIPT 4 101
processes but these transitioned to a column configuration and full automation as a miniaturized
102
technique. Despite the advantages of SPAD, there remains the critique that large excess of reagents
104
are required. While these are often chromatographically well separated from the derivatives, the
105
excess reagent can contribute contaminants with potential for interference to analysis. Excess of
106
reagent may be required to achieve complete reaction and require cleanup protocols. The
107
reagent excess can compromise the reusability of the solid phase – for instance shortening the
108
life time of the SPME fiber. Use of reagents also precludes direct in-vivo sampling. However,
109
extraction followed by derivatization of the sorbed analyte can provide advantages of SPAD
110
without exposing the organism to reactive compounds.
112 113
SC
This review covers microextraction applications of SPAD with a focus on literature
M AN U
111
RI PT
103
published 2017-2018.
2. Solid phase analytical derivatization early developments
The XAD resins formulated as beads were widely used in SPE applications due to their
115
high surface area and high absorptivity of lipophilic analytes and reagents mainly through
116
hydrophobic bonding and concentrating a smaller volume of beads. In addition, the XAD resins
117
are stable to acidic and alkaline. The first SPAD reaction reported in 1984 as well as subsequent
118
methods used XAD-2, a styrene–divinylbenzene crosslinked polymeric macroreticular resin as
119
the solid phase [6]. Use of XAD resins with different physical or chemical characteristics did
120
not improve yield or reaction rate.
TE D
114
A variety of reagents and analytes were tested. Pentafluorobenzyl bromide (PFBBr)
122
served to form PFB esters of carboxylic acids or PFB ethers of phenols such as
123
tetrahydrocannabinol and other cannabinoids [1-3]. Derivatization at alkaline pH using PFBBr
124
produced PFB esters of carboxylic acids and PFB ethers of phenols. At neutral pH the SPAD
125
yielded only the esters of carboxylic acids. Addition of small volumes of C1-C5 alcohols to a
126
SPAD reaction at alkaline pH suppressed the formation of the esters but not of phenols thus
127
providing enhanced specificity to the analysis. Derivatization with pentafluoropyridine (PFP)
128
under alkaline SPAD conditions was selective towards phenols in the presence of carboxylic
129
acids. Pentafluorobenzyl hydroxyl amine (PFBONH2), benzyl hydroxylamine (BzONH2) rapidly
130
form PFBoxime or Bzoximes of aldehydes and ketones ranging in size from C1-C20.
AC C
EP
121
Under
ACCEPTED MANUSCRIPT 5 131
acidic SPAD 2,4 dinitrophenylhydrazine (DNPH) rapidly formed the hydrazone product of both
132
aldehydes and ketones. In recent years researchers have explored novel solid phases for SPAD. These included
134
solid phase microextraction fibers, various nanoparticles, monolith columns, metals, metal
135
oxides, glass plates, GC injection ports, hollow fibers, stir bars and ionic liquids. Some of the
136
key milestones in SPAD method development are listed in Table 01.
137