Patent Application
20070281361
The determination of the concentrations free amino acids in biological samples is a very important clinical diagnostic test. Most of the clinical laboratories perform the assay using the High Performance Liquid Chromatographic (HPLC) technique. There are only two commercial HPLC methods that are widely in use for the past three decades. They are the ion exchange method (about 6 vendors are there around the world) and the “PICO Tag™” method (1) of “Waters” chromatographic company USA. Both methods are Ultraviolet detection methods. The two methods represent two different types of HPLC methods. The ion-exchange method is a post column derivatization method and the PICO tag method is a pre column derivatization method which uses Phenylisothiocyanate (PITC) as the derivatizing agent. The present inventor published his first paper on the separation of amino acids using PITC in1993 (2)
For more than 50 years the clinical amino acid analysis of biological samples has been a problem and does not serve well clinical needs because of the long run times of 160 to 300 minutes per sample. Ion-exchange method is more widely used in clinical laboratories. Many of these laboratories still have the old Beckman ion exchange instrument (Beckman stopped the production of the instrument 20 years ago) and their run time per sample is 300 minutes. Recent commercial ion exchange method has a run time of 160 minutes per sample. Present commercial and in house HPLC methods have many other problems—the derivatization methods are complex, the derivatizing reagents and the derivatives are unstable, derivatization is not comprehensive, the instrumentation is sophisticated and very expensive, mobile phases are complex, hard to prepare, and also very expensive, and the complex gradient and column heating programs render trouble shooting peak resolutions very difficult. Two other major drawbacks of the present HPLC methods are 1) they can not provide a comprehensive amino acids profile using blood spot (critical for new born babies) and 2) do not estimate the total and or free concentrations of the two important thiol amino acids—Homocysteine and Cysteine as part of the profile. Although the two commercial methods—the ion-exchange method and PICO TAG method are “mature” and in use for more than 30 years the tests are very difficult to perform and highly skilled work force is a necessary. There is a lack of competition in the field for many decades and the cost of the assay is skyrocketing around the world. Six years ago the cost of one ion-exchange column was $3000 and in 2006 it was $5000. The present methods are uneconomical and drain our valuable resources—time and money. In 1993 “Waters” introduced a novel pre column derivatizing reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (ACCQ TAG™) that reacts fast and in an elegant manner with amino acids (3) but it is a failure in the separation of all important amino acids in physiological samples. Little has happened in the last 14 years in spite of the innumerable varieties of analytical columns introduced by “Waters” to change the situation. The combined physical and chemical properties of the derivatives of amino acids in biological samples with ACCQ TAG are not amenable for the separation of important 27 amino acids. There has not been any break through in simplicity, sensitivity, robustness, and many other ideal features necessary for this clinical test to be successful for a long time. The test is a necessity but it is a big drag in a modern clinical laboratory.
It is well known that the best and simplest method for the analysis of amino acids is the pre-column derivatization method because of the short run times and ease of peak resolutions using reversed phase columns with high theoreretical plates. An ideal pre-column derivatizing agent should have the following characteristics: It is a stable compound, readily available, quickly (2-5 mins) reacts with amino acids (both primary and secondary) and forms only one derivative with each and every one of them, the derivatives are stable in solution, imparts good (femto mole) sensitivity for the assay, the derivatives have the optimal hydrophilic and hydrophobic characteristics to be amenable for the separation of all the important amino acids in a short time, and the derivatives are readily purified from excess reagent and side products of the reaction. Such a derivatizing agent (a fluorescent tag) was initially conceived more than 30 years ago by Dr. Perrett in England and it has been rediscovered by the present inventor and nicely exploited and proven to be the best for the analysis of amino acids profile in biological samples and it is the principal ingredient of this Patent application.
The reagent 1-Napthylisocyanate (NIC), was first used Amos Neidle and his co workers in 1989 (4).for the separation of 21 amino acids in 160 minutes (in addition there was a recycle time of 40 minutes before next injection). They barely recognized the importance and properties of the reagent NIC. The HPLC instrument used by them was quite antiquated and it lacked any modem features. The separation of the amino acid derivatives was not state of the art work even at the time. The work lacked precision and accuracy data on human plasma samples and normal values for healthy volunteers. Neither the original workers nor others who pursued work in the area recognized the great properties of the fluorescent tag and its amino acids derivatives until almost 20 years later by the present inventor.
Sodium dihydrogen phosphate, Sodium Hydroxide, Boric acid, Perchloric acid, Tris(carboxy ethylphosphine Hydrochloride), HPLC solvents acetonitrile, methanol, and water and Sodium heptane sulfonate were purchased from Fisher Scientific, Fair Lawn N.J. Amino acid standards (solids) were obtained from Sigma, St Louis, Mo.
1.0 M Borate buffer was prepared from boric acid and the pH was adjusted to 6.25 using sodium hydroxide solution.
Amino acid Standard: 400 μM/L standard was prepared by dissolving 0.1 mM amount of each amino acid (except—Homocystine and Cystine—only 0.05mM) were weighed and transferred to a 250 ml beaker, The amino acids were dissolved using minimum amount of 1M Sodium hydroxide solution and neutralized (using a pH meter and 1M hydrochloric acid solution). The neutral solution was transferred quantitatively to a 250 ml volumetric flask, the solution made up to volume and mixed well for uniform concentration. The solution was aliquoted and kept frozen in −80° C. freezer. The solution contained all amino acids including Glutamine, Glutamic acid, Aspartic acid, Aspargine and Tryptophan and was found to be stable for at least six months. The standard solution compared well with several of our own separate preparations and against external standard from Sigma Chemical Company.
Internal Standard: Both Nor Leucine and Nor Valine were found to be good for the work. 250 μM/L neutral solutions were separately prepared and aliquots were kept frozen at −80° C. Norvaline was used when Cystathionine had to be quantitated, Cystathionine elutes near Norleucine in our conditions of analysis.
Derivatization reagent: It was prepared by dissolving 10 μl of 1-Naphthylisocyanate in 4 ml of dry acetone in a test tube. The solution was prepared just prior to use.
The Hitachi HPLC system consisted of two (Model L2130) pumps, a built in Model L2200) degasser, an Auto sampler with Peltier cooling, a (Model L2300) Column oven, and a (Model L2480) Fluorescence detector with a 3 μl flow cell and a Dell Optiplex GX270T computer with EZchrom Elite software for data processing and control of all the instruments. The analytical column is a 3 micron C-18 ODS2, Hypersil column 15×0.46 cm (from Fisher-Thermo Electron, Bellefonte, Pa.)
A microcentrifuge (Eppendorf model 5418) was obtained from Fisher-Thermo Electron.
A. For plasma or Aqueous Standard
Pipetted 100 μl of water (blank) or aqueous standard or protein free filtrate (prepared using EDTA plasma and “Micron YM-10” filter device from Millipore, Mass.) into a 2 ml capacity labeled micro centrifuge tube with caps. To all tubes, 100 μl of internal standard, (Norleucine) 50 μl of borate buffer and 300 μl of HPLC grade water were added. The solutions in the centrifuge tube were gently mixed. 250 μl of derivatization reagent was added to each tube. Immediately after addition the tube was closed and the solution vortexed for few seconds. After a minute, the fine precipitate of 1,1′-dinaphthyl urea was filtered using a Whatman filter device (Mini UniPrep™ 1-5 ml volume, with nylon membrane, pore-2 μm) and the filtrate was extracted with 1 ml of Cyclohexane (to remove excess reagent and its hydrolytic product 1-Naphthylamine) and the top organic layer was removed under reduced pressure. Cyclohexane wash was repeated twice more and the aqueous layer stored at −4° C. until analysis. Analysis was performed diluting 40 μl of the solution with 200 μl of HPLC water and 3 μl injected into the column. Chromatograms of an aqueous standards, healthy volunteer plasma and reagent blank are shown in
Alternative method for protein free filtrate: When 100 or 200 μl of plasma only was available, added corresponding amount of internal standard and equal volume (100 or 200 μl) of 6% perchloric acid to the plasma. The solutions were immediately mixed, left to stand for 5 minutes and then centrifuged. 200 or 300 μl of clear supernatant was transferred to a new micro centrifuge tube, neutralized and used for derivatization.
B. For Blood Spot
The derivatization procedure for the blood spots was similar to that for plasma. Two blood spots were obtained using ⅛th inch diameter hand puncher. Amino acids were extracted by gentle tumbling using an aqueous organic mixture of 200 μl of methanol and 50 μl of an aqueous solution of internal standard (62.5 μM) solution) for 45 minutes using a 2 ml micro centrifuge tube. 150 μl of the top clear solution was transferred to a 13×75 mm glass test tube and the solution evaporated at 30° C. (oven) for 30 minutes. The residue was dissolved in 300 μl of HPLC water and treated with 25 μl of 1M borate buffer and 100 μl of the derivatizing reagent (which was obtained by diluting 1:4 the initial solution (refer above to derivatizing reagents preparation) with acetone. The standard for the blood spot work was processed as follows. Pipetted 3 μl of 400 μM/L aqueous standard solution used in the regular plasma work into a 2 ml micro centrifuge tube containing 200 μl of HPLC water and 50 μl of (62.5 μM/L) internal standard. The solutions were mixed and 150 μl of the solution was transferred to 13×75 mm glass test tube. To the standard solution, added 25 μl of 1M borate buffer (pH 6.25) 200 μl of HPLC water, and 100 μl of derivatizing reagent (refer above). After standing at room temperature for a minute with the derivatizing reagent the blood spot or the standard solution was centrifuged or filtered to remove the white precipitate and the clear top solution was transferred to new micro centrifuge tube (2 ml) and extracted 3 times with 0.5 ml cyclohexane (the top layer aspirated under reduced pressure). 3 μl of the derivatized solution was injected into the HPLC column. Chromatograms of an aqueous standard and a CDC blood spot control are shown in
Mobile phase (MP) A is a mixture of 20 mM phosphate buffer pH 5.9 and methanol 94:6 V/V containing 15 mg Sodium heptane sulfonate per 100 ml of solution.
Mobile phase (MP) B is a mixture of 20 mM phosphate buffer pH 5.73, methanol and acetonitrile 50:24:26 V/V containing 15 mg of Sodium heptane sulfonate per 100 ml of solution.
The column was equilibrated at initial conditions for 10 minutes (cycle time) before next injection. Calculations were based on peak height determinations through out work
Based on the chromatogram of a derivatized aqueous standard of Amino acids for physiological samples the relative fluorescence response of all important amino acid derivatives were calculated using Valine peak height as the reference. The values are given in Table 1. The highest responses were noted for Aspartic acid, Glutamic acid and Alanine. The smallest responses were noted for Cystine and Tryptophan. The latter two derivatives have two Naphthyl carbamoyl groups per molecule and internal quenching decreases their fluorescence response considerably. Although such quenching occurs in Lysine and Ornithine, their fluorescence response is good and better than those for Cystine and Tryptophan and the former amino acids can be estimated by fluorescence detection. Except for the few above mentioned all other amino acids have approximately the same fluorescence responses
The smallest amount of sample and standard was used in the blood spot work. CDC has determined that the volume of plasma in two blood spots of ⅛th inch diameter manual punch is 3.0 (±0.2%) μl. The final volume of derivatized solution in the blood spot (and the corresponding aqueous standard) work was about 300 μl and 3 μl was injected into the column.
The smallest concentration of aqueous standard used was 10 μM/L. The final volume of the derivatized solution was about 300 μl. 1 pico mole of each standard was injected into the column. Using the standard chromatogram and a signal to noise ratio of 3:1, the detection limits for the amino acids ranged from 40-950 femto moles. The smallest detection limit (40 femto moles) was calculated for Aspartic acid, Glutamine and Alanine and the highest detection limit (980 f moles) was noted for Tryptophan and the next higher detection limit was for Cystine.
More than 40 years since the beginning of amino acids profile analysis for clinical use, 1-Napthylisocyanate (NIC) has been found and proven as the best pre column derivatizing reagent. The fluorescent tag reacts quantitatively in one minute with all amino acids, and forms only one derivative with each one of the amino acids. The derivatives are very stable in solution for more than a day and longer (months/years) at lower temperatures (−4 to −80° C.). 35 amino acids (plus two internal standards and ammonia) in plasma have been separated in record shortest time of 20 minutes using a simple high pressure binary gradient, reversed phase (C-18, 3 micron, 150×4.6 mm) column, and a femto moles sensitive method. The challenge was accomplished with great success by optimization of several factors. The robustness of the method was established from linearity (0 to 1200 μM/L), precision (<6%), and accuracy (95%) studies and determination of plasma concentrations of free amino acids for few healthy volunteers and abnormal patients. For the first time a sample can be analyzed with in one hour of its arrival in the laboratory. 48 samples can be analyzed in 24 hours using one ($42,000) instrument. The method has two more unique, experimentally confirmed features unknown with any of the present amino acids profile methods. It provides: 1) a comprehensive amino acids (27) profile using a blood spot (3 μl of human plasma, the work was validated using “CDC” blood spot controls for two years) and 2) the free and total concentrations of Homocysteine and Cysteine as part of a full amino acids profile on human plasma. The following simple features of the method: 1 mobile phases, 2 traditional high pressure binary gradient system, 3 gradient program, 4 analytical column, 5 adaptation, 6 trouble shooting peak resolutions, 7 femto mole sensitivity, 8 very low cost of the instrument, column and reagents, 9 less than 20 minutes run time for a sample without temperature variations, 10 good precision (<6%) and accuracy (95%) of the method, 11 full plasma amino acid profile using one blood spot, 12 plasma amino acid profile that will include free and total concentrations of Homocysteine and Cysteine—qualify the method as the best choice for amino acids profile in biological samples and protein hydrolyzates in many decades. The method will lower health care costs by more than 60% in the area and a savings of at least $50 million (US) per year in the world. The method provides patient results in the shortest time in the history of the method and ensures quality patient care. The attributes of the method are due to the versatility of the derivatizing agent, properties of its amino acid derivatives and optimization of factors that influence their separation and detection.
Values are for the same concentration of aqueous standard (except for NVAL and NLEU)
*1-Naphthylisocyanate
**PITC—Phenylisothiocyanate,
***OPA—orthoPhthalaldehyde,
****uses Ninhydrin -coloring reagent
¶3 μl plasma.
§First time introductions in the history of the method
$ACN); MP-B- 55:45 buffer:ACN
@20, omitted-OHPRO, CIT, ASN, TAU
§MP—mobile phase,
*refer Table 4 for abbreviations,
$ACN Acetonitrile,
@Authors didn't have a reliable standard.
¶Historical features
The retention times of peaks vary with different column lots, mobile phase composition, gradient etc., but the order of elution is the same.
¶Internal Standard. All the 40 amino acids can be separated by the gradient method used in this work.
*N = 6
36 ± 2.3
304 ± 20.5
31 ± 1.8
38 ± 2.2
66 ± 2.9
76 ± 4.7
72 ± 2.6
*n = 16
• N = 8 (concentrations added ranged from 25 to 400 μM/L)
*Adults ages 20-70
¶n = 12
N = 5 Intra assay
Data for the Chromatogram of Normal Patient Plasma—
Data for the Chromatogram of CDC Blood Spot Standard—
Data for the Chromatogram of CDC Blood Spot Quality Control—
Total number of Figures, is 10. For abbreviations please refer Table 4, the Y axis in all figures corresponds to micro volts—mv and X axis corresponds to time in minutes
The chromatogram shows the separation of all important amino acids (27+ammonia) in an aqueous neutral standard solution. It highlights the following good features: 1 only one derivative is formed for each amino acid, 2 the Naphthyl carbamoyl derivatives have the best properties for separation, 3 proves the good experimental conditions for their separation and 4 an idea of the fluorescence response of different amino acids derivatives. The figure is historical because the separation is good, comprehensive and fastest in the history. The concentration of all amino acids is 400 μM/L, except PSER & AABU (25 μM/L each), NLEU—the internal standard (250 μM/L), (CYS)2—200 μM/L and AMMO—is an impurity.
The figure shows the separation of an aqueous neutral solution of 35 amino acids (and AMMO) in record short time of about 20 minutes.
Shows the Fluorescence chromatogram of a healthy adult. The two peaks at 13.02 and 13.56 minutes are unidentified peaks. Analysis report for the sample is on page 42.
Shows the fluorescent chromatogram of blank reagent using water (instead of sample or standard) and internal standard. It is a nice reflection of the absence of any interference from reagents in the assay.
Shows the chromatogram of our aqueous standard (400 μM/L) for blood spot amino acids profile using 3 μl of the solution. This old standard used was prepared in 0.1 normal hydrochloric acid. The concentrations of GLN and GLU are not 400 μM/L. GLN is hydrolyzed in acid medium (0.1M HCl) to GLU. The standard was used for the estimation of amino acids other than GLN, GLU, ASN, ASP and TRP in CDC quality control blood spots. Analysis report for the standard against another 400 μM/L standard is given on page 46
Shows the Chromatogram of a CDC blood spot quality control sample. The figure shows the nice separation of 27 amino acids, ammonia, and the internal standard NLEU. This figure shows the estimation of all important amino acids using 3 μl of plasma sample. The total volume of the derivatized solution will be about 250-300 μl, of which 3 μl is injected into the column with the detector photo multiplier set at the lowest voltage setting. The analysis report for the sample is given on page 47.
This is the first fluorescence chromatogram of aqueous amino acids profile standard (400 μm/L) with peaks for “free” Cysteine (CYS) and Homocysteine (HCY) and has a run time of about 20 minutes. (Refer claim 5). The retention time for CYS is 9.2 min and that for HCY is 11.1 min. In this Fig CYS and AABU peaks are some what close due to the high concentration of AABU (400 μM/L). Normal concentration range for AABU for healthy person is 15-28 μM/L. This figure illustrates two important facts: 1) 1-Napthylisocyanate is the best pre column derivatizing agent for human plasma amino acids profile, because it facilitates the simple reduction of the preformed derivatives of Cystine and Homocystine and 2) our carefully optimized separation and detection experimental conditions are one of the best and they facilitate the appearance CYS and HCY peaks in very nice positions in the chromatogram. The profile chromatogram is the best in the clinical amino acids analyses history.
It is the first free amino acids profile chromatogram of a normal patient's plasma sample with peaks for free Cysteine (CYS) and Homocysteine (HCY). (Refer claim 5) with a run time of about 20 minutes. The two peaks appear in positions where there is no interference from other amino acids in plasma. The figure illustrates the versatility and superiority of the derivatizing agent NIC and its amino acids derivatives and our carefully optimized chromatographic conditions for the profile analysis.
It is the fluorescence chromatogram of patient plasma with abnormal Citrulline concentration. Citrullineamia patient. The main peak of interest in this Figure is the CIT peak.
It is the fluorescence chromatogram of a patient with abnormal Arginine concentration. Arginase enzyme deficiency patient. The main peak of interest in this Fig is the ARG peak
All FIGS. from 2 to 9 are replacement figures.
New
New
| Number | Date | Country | |
|---|---|---|---|
| 60799401 | May 2006 | US |
