	The building blocks of peptides and proteins, amino acids, are present in animals, humans, and plants. The analysis of amino acids is of essential significance in several areas including food science, clinical diagnostics, and pharmaceutical products research. Classical liquid chromatography (LC) determination of amino acids is performed with sample pretreatment including pre-column as well as post-column derivatization to improve or enable spectroscopic detection.

	Various methods have been devised for the pre-column derivatization of amino acids. One of the most established methods is based on the reaction of o-phthaldialdehyde (OPA) with primary amines at basic conditions as shown in Figure 1 (1). The addition of mercaptopropionic acid (MPA) to the reaction solution increases the polarity of the derivatized products, reducing retention of the analytes on a reversed phase column and resulting in improved separation.

This work shows the application of a fully automated, rapid, and accurate amino acids pre-column derivatization method using the autosampler pretreatment function of the high performance liquid chromatography (HPLC) system. After derivatization, amino acids were separated on a C18 column and determined by fluorescence detection. The subsequent hyphenation to mass spectrometry (MS) was performed for peak tracking in real samples.

 Ultrapure water (ASTM type 1) was prepared with a water purification system by Sartorius. Methanol and acetonitrile were of LC–MS quality (Honeywell). 3-mercaptopropionic acid (≥ 99%), boric acid (≥ 99.5%), and hydrochloric acid (≥ 37%, puriss. p.a.) were purchased from Sigma Aldrich. Formic acid (reagent grade, ≥ 95%), sodium hydroxide solution (10 M, Ultra), ammonium bicarbonate (≥ 99.5%), and o-phthaldialdehyde (≥ 98.5) were sourced from Fluka. L-amino acids standard solution (2.5 mM in 0.1 M HCl), γ-aminobutyric acid (≥ 95%), and L-glutamine (≥ 95%) were from Sigma Aldrich. DL-asparagine and DL-tryptophan (≥ 99%, respectively) were from Sigma Chemical Company.

Preparation of Mobile Phase, Derivatization Reagents, Standards, and Samples:

 Mobile phase A was prepared by dissolution of ammonium bicarbonate in water (20 mM) and adjusted with formic acid to pH 6.9. A 0.2 M borate buffer was prepared by dissolution of boric acid in water and adjusted to pH 9.2 with 10 M NaOH. MPA (20 µL) and OPA (20 mg) were dissolved in 10 mL of the prepared borate buffer, respectively. All derivatization regents were stored at 5 °C for a maximum of 1 week prior to analysis. A 100 µM amino acid standard was obtained by dilution of a commercially available mixture of amino acids or 2.5 mM stock solutions, which were prepared from γ-aminobutyric acid, glutamine, asparagine, and tryptophan in 0.1 M hydrochloric acid. A white wine sample was injected into the HPLC system after filtration with a 0.2-µm pore size filter.

 The derivatization procedure is presented in Figure 2. A 10-µL amino acid standard solution or sample was transmitted to a 1.5 mL vial with a 200 µL glass inlet. A 45-µL mixture of MPA and OPA solution (1/1) was added, followed by addition of 45 µL of 0.2 M borate buffer. After mixing (3 × 40 µL) and a waiting time of 1 min, 1 µL of the mixture was injected for HPLC analysis.

 The HPLC system was equipped with a fluorescence detector, followed by a single quadrupole mass spectrometer. Analytical conditions are presented in Table 1.

	A total of 19 amino acids could be determined using a newly developed, automated pretreatment program for derivatization (Figure 3). The information provided by retention times as well as mass-to-charge ration (

) measured was applied for the determination of amino acids in a white wine sample (Figure 3 and Table 2).

	A fully automated derivatization method for amino acid analysis using the pretreatment function of the HPLC autosampler was developed. The method was applied successfully in the determination of amino acids in a white wine sample. This time-saving approach of a fully automated derivatization method, in combination with fluorescence and MS detection, is a valuable tool for laboratories requiring high throughput analysis, while also reducing the risk of human error during sample preparation.


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Articles

Food and Beverage

The building blocks of peptides and proteins, amino acids, are present in animals, humans, and plants. The analysis of amino acids is of essential significance in several areas including food science, clinical diagnostics, and pharmaceutical products research. Classical liquid chromatography (LC) determination of amino acids is performed with sample pretreatment including pre-column as well as post-column derivatization to improve or enable spectroscopic detection.

Fast and Sensitive Analysis of Amino Acids Using Automated Pre-Column Derivatization

	UCT’s Refine™ Ultra-Filtration Technology allows for sample precipitation and filtration to occur simultaneously in the individual column or well. A novel, hydrophobically treated, submicrometer frit combination facilitates the removal of sample proteins without a complicated extraction. 

	a) Place a collection plate under the Refine™ Ultra-Filtration Plate.
	b) Add 250 µL of urine and 250 µL Abalonase Ultra Enzyme working solution.
	c) Hydrolyze for 30 min at 55°.
	d) Further dilute sample by adding 500 µL D.I. H

O.
	e) Mix the sample. This can be executed via vortexing at maximum speed or multiple pipette aspirations and dispenses.
	f) Filter the sample using one of the following techniques:

	1. Centrifuge: For 5 min at 500 g or until filtrate is collected.
	2. Vacuum: Apply vacuum at ~20” of Hg for up to 5 min or until filtrate is collected.
	3. Positive pressure: Apply 2–5 psi using positive pressure for up to 5 min or until filtrate is collected.

 Agilent™ 1200 HPLC and AB Sciex™ 4000 Q Trap (MS/MS)
	

 100 × 2.1 mm, 3-μm UCT Selectra® DA HPLC column
	

 10 × 2.1, 3-μm UCT Selectra® DA guard column 
	

	Using the above procedure, the Refine™ Ultra-Filtration technology allowed for a reduction in instrumental back pressure and removal of unwanted matrix components in urine samples. Overall, this allows for an end user to have enhanced analyte selectivity and an increase in HPLC and UHPLC column life.


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Medical/Biological

UCT’s Refine™ Ultra-Filtration Technology allows for sample precipitation and filtration to occur simultaneously in the individual column or well. A novel, hydrophobically treated, submicrometer frit combination facilitates the removal of sample proteins without a complicated extraction. 

In-Well Hydrolysis Using UCT’s Refine™ Ultra-Filtration Technology

Abuse of synthetic opioid prescription painkillers such as fentanyl, along with a rapidly growing list of illicit analogues, is a significant public health problem. In this study, we developed a simple dilute-and-shoot method that provides a fast 3.5-min analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC–MS/MS using a Raptor Biphenyl column.

	In recent years, the illicit use of synthetic opioids has skyrocketed, and communities worldwide are now dealing with an ongoing epidemic. Of the thousands of synthetic opioid overdose deaths per year, most are related to fentanyl and its analogues. With their very high analgesic properties, synthetic opioid drugs such as fentanyl, alfentanil, remifentanil, and sufentanil are potent painkillers that have valid medical applications; however, they are also extremely addictive and are targets for abuse. In addition to abuse of these prescription drugs, the current opioid crisis is fueled by a growing number of illicit analogues, such as acetyl fentanyl and butyryl fentanyl, which have been designed specifically to evade prosecution by drug enforcement agencies.
	
	As the number of opioid drugs and deaths increases, so does the need for a fast, accurate method for the simultaneous analysis of fentanyl and its analogues. Therefore, we developed this LC–MS/MS method for measuring fentanyl, six analogues, and one metabolite (norfentanyl) in human urine. A simple diluteâandâshoot sample preparation procedure was coupled with a fast (3.5 min) chromatographic analysis using a Raptor Biphenyl column. This method provides accurate, precise identification and quantitation of fentanyl and related compounds, making it suitable for a variety of testing applications, including clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.
	
	

The analytes were fortified into pooled human urine. An 80 µL urine aliquot was mixed with 320 µL of 70:30 water–methanol solution (fivefold dilution) and 10 µL of internal standard (40 ng/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat. #25895). After filtering through the 0.2 µm PVDF membrane, 5 µL was injected into the LC–MS/MS.
	
	

Calibration Standards and Quality Control Samples: 

The calibration standards were prepared in pooled human urine at 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.0, 25.0, and 50.0 ng/mL. Three levels of QC samples (0.75, 4.0, and 20 ng/mL) were prepared in urine for testing accuracy and precision with established calibration standard curves. Recovery analyses were performed on three different days. All standards and QC samples were subjected to the sample preparation procedure described.
	
	LC–MS/MS analysis of fentanyl and its analogues was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows, and analyte transitions are provided in Table 1.

Analytical column: Raptor Biphenyl (5 µm, 50 mm × 2.1 mm; cat. #9309552)
	Guard column: Raptor Biphenyl EXP guard column cartridge, (5 µm, 5 mm × 2.1 mm; cat. #930950252)
	Mobile phase A: 0.1% Formic acid in water
	Mobile phase B: 0.1% Formic acid in methanol
	Gradient        Time (min)         %B
	                      0.00                     30
	                      2.50                     70
	                      2.51                     30
	                      3.50                     30
	Flow rate: 0.4 mL/min
	Injection volume: 5 µL
	Column temp.: 40 °C
	Ion mode: Positive ESI
	
	

 All eight analytes were well separated within a 2.5-min gradient elution (3.5-min total analysis time) on a Raptor Biphenyl column (Figure 1). No significant matrix interference was observed to negatively affect quantification of the fivefold diluted urine samples. The 5-µm particle Raptor Biphenyl column used here is a superficially porous particle (SPP) column. It was selected for this method in part because it provides similar performance to a smaller particle size fully porous particle (FPP) column, but it generates less system back pressure.

Linear responses were obtained for all compounds and the calibration ranges encompassed typical concentration levels monitored for both research and abuse. Using 1/x weighted linear regression (1/x

 for butyryl fentanyl), calibration linearity ranged from 0.05 to 50 ng/mL for fentanyl, alfentanil, acetyl fentanyl, butyryl fentanyl, and sufentanil; from 0.10 to 50 ng/mL for remifentanil; and from 0.25 to 50 ng/mL for norfentanyl and carfentanil. All analytes showed acceptable linearity with r2 values of 0.996 or greater and deviations of <12% (<20% for the lowest concentrated standard).
	
	

 Based on three independent experiments conducted on multiple days, method accuracy for the analysis of fentanyl and its analogues was demonstrated by the %recovery values, which were within 10% of the nominal concentration for all compounds at all QC levels. The %RSD range was 0.5–8.3% and 3.4–8.4% for intraday and interday comparisons, respectively, indicating acceptable method precision (Table 2).


	
	A simple dilute-and-shoot method was developed for the quantitative analysis of fentanyl and its analogues in human urine. The analytical method was demonstrated to be fast, rugged, and sensitive with acceptable accuracy and precision for urine sample analysis. The Raptor Biphenyl column is well suited for the analysis of these synthetic opioid compounds and this method can be applied to clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.


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Analysis of Fentanyl and Its Analogues in Human Urine  by LC–MS/MS

Intrinsically-disordered proteins cannot be analyzed by SEC column calibration. SEC-MALS provides accurate molecular masses of these proteins and of their complexes with other proteins.

	The S100 family is a group of dimeric, calcium-binding proteins which have been found to be overexpressed in several types of cancer. S100 proteins bind to the C-terminal region (293-393) of p53, the most critical tumour suppressor involved in apoptosis, cell cycle arrest, and DNA repair. Since S100 is dimeric and the C-terminus of p53 encompasses a tetramerization domain, the potential range of complexes incorporating p53 and S100 is large.

	The p53 variants have a large fraction of intrinsically disordered structure and do not elute according to globular protein standards in size-exclusion chromatography (SEC). However, multi-angle light scattering (MALS) determines molecular mass from first principles. SEC-MALS determines the molecular weight of the complexes independently of protein standards. We utilized SEC-MALS to investigate how S100 proteins bind to p53 in its different oligomeric states, using monomeric (L344P) and dimeric (L344A) p53 mutants.

	The experimental system consisted of an HPLC and SEC column plumbed in series with a DAWN MALS detector (Wyatt Technology) and an Optilab differential refractive index (dRI) detector (Wyatt Technology).

	The p53 mutants and S100 proteins were first characterized separately by SEC-MALS to determine their molecular masses and homogeneity. They were then incubated at various stoichiometric ratios-4:1, 2:1, 1:1, 1:2, and 1:4-and analyzed on the SECâMALS system.

	Molar mass results of the individual p53 proteins are overlaid with the light scattering (solid lines) and dRI chromatograms (dotted lines) in Figure 1. The molar masses correspond to a monomer of 11.2 kDa for the L344P variant, a dimer for the L344A variant, and a tetramer for p53 wild-type.

Chromatograms and molar masses for the pre-incubated mixtures are presented in Figure 2. Monomeric p53 (293-393) L344P (11.2 kDa) and dimeric S100B (21.4 kDa) both eluted with a retention time of ~17.5 min.

The complex molar mass of ~32 kDa at ~16 min is independent of the relative ratio of the two proteins, indicating that one dimer of S100B binds only one monomer of p53. No complexes consisting of a dimer of S100B and two monomers of p53 (~43 kDa) could be detected. In addition, S100B was able to disrupt the dimeric p53 (293-393) L344A mutant and form a complex with the monomer.

	Interestingly, SEC-MALS revealed that S100B was also able to form a stable complex with tetrameric p53 (293-393) consisting of 4 dimers of S100B bound to a tetramer of p53.

	SEC-MALS helped understand how S100 proteins influence the oligomerization of p53.  Based on these results and in vivo studies, we established a binding model where S100 proteins can activate tetrameric p53 but inhibit p53 activity binding to the monomer by shifting the tetramerization equilibrium. SEC-MALS is a valuable tool in structural biology for studying the stoichiometry and formation of many types of protein-protein complexes.


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Stoichiometry of Intrinsically-Disordered Protein Complexes

Drug-antibody ratio (DAR) is a critical attribute of antibody–drug conjugates (ADCs). DAR may often be determined quickly and effectively by combining SEC with three online detectors: MALS, UV, and dRI.

	There has been a significant resurgence in the development of antibody–drug conjugates (ADC) as target-directed therapeutic agents for cancer treatment. Among the factors critical to effective ADC design is the drug-antibody ratio (DAR), which describes the degree of drug appended to the antibody and directly impacts both potency and potential toxicity of the therapeutic. In addition to therapeutic and physiological effects, DAR can also affect drug product properties such as stability and aggregation. Determination of DAR is, therefore, of critical importance in the development of novel ADC therapeutics.

	DAR is typically assessed by mass spectrometry (MALDI-TOF or ESI-MS) or UV spectroscopy, both of which are subject to certain limitations: 1) calculations based on UV absorption are often complicated by similarities in extinction coefficients of the antibody and small molecule; 2) mass spectrometry, though a powerful tool for molecular weight determination, depends on uniform ionization and recovery between compounds-which is not always the case for ADCs. We present here a method for DAR determination based on SEC-MALS in conjunction with UV absorption and differential refractive index (dRI) detection, which overcomes these limitations for many ADCs.

	Two model ADCs based on the same monoclonal antibody and drugâlinker system, but different conjugation processes, were analyzed. In this example the antibody has been alkylated with a compound having a nominal molar mass of 1250 g/mol.

	Each ADC was passed through size-exclusion chromatography (SEC) followed by the HPLC’s UV detector, a DAWN multi-angle light-scattering (MALS) detector (Wyatt Technology) and an Optilab differential refractive index (dRI) detector (Wyatt Technology). Protein molar mass (M

) of the ADC were determined at each elution volume by analysis of signals from the three detectors.

	Data collection and analysis were performed in the ASTRA software (Wyatt Technology) using ASTRA’s Protein Conjugate Analysis method. Conjugate analysis is automated within ASTRA using the differential refractive index increments (

) and UV extinction coefficients, which are determined empirically for each species or mined from the literature.

	Figure 1 shows UV traces for the two model ADCs along with the calculated values of M

 of each. As expected, the molar masses of the antibody components are quite similar; but Mdrug differs considerably. Despite the drug constituting just a small fraction of overall molar mass, this highly sensitive system shows robust Mdrug values which are well-distinguished from each other and vary little over the peak. The inset presents just Mcomplex, clearly showing the sensitivity of the system which differentiates the two molecules quite well.

Final DAR is calculated from the ratio of M

 to the molar mass of a single drug + linker compound, shown in the table. The results are consistent with values obtained by orthogonal techniques. Note that the molar mass traces for the conjugated moiety represent the total amount of attached pendant groups; the horizontal trends indicate that modification is nearly uniform throughout the population eluting in that peak.

	This analysis was successful thanks to the relatively large total appended mass, about 5% (ADC2) to 8% (ADC1) of the total molar mass of the complex. In general, for accurate SEC-MALS-UV-dRI protein conjugate analysis, the modifier mass should be at least 3% of the total. Hence a lower degree of conjugation might not have been amenable to analysis by this method.

	SEC-MALS-UV-dRI provides an easy, accurate means to determine DAR of ADCs in the course of product development and quality control. It is suitable for relatively high DAR values where the total drugâlinker mass is at least 3% of the overall complex mass.

The Application Notebook-07-01-2018

Fast and Sensitive Analysis of Amino Acids Using Automated Pre-Column Derivatization

In-Well Hydrolysis Using UCT’s Refine™ Ultra-Filtration Technology

Analysis of Fentanyl and Its Analogues in Human Urine by LC–MS/MS

Stoichiometry of Intrinsically-Disordered Protein Complexes

Drug-Antibody Ratio of an Antibody–Drug Conjugate by SEC-MALS