CN116338048A

CN116338048A - Analysis method for simultaneously detecting multiple proteins in urine

Info

Publication number: CN116338048A
Application number: CN202310310256.1A
Authority: CN
Inventors: 贡文秀; 黄学英
Original assignee: Suzhou Saifen Technology Co ltd
Current assignee: Suzhou Saifen Technology Co ltd
Priority date: 2023-03-28
Filing date: 2023-03-28
Publication date: 2023-06-27

Abstract

The invention provides an analysis method for simultaneously detecting multiple proteins in urine, which comprises the steps of filtering the urine by a hydrophilic MCE filter membrane, and sequentially subjecting the urine to a volume column gel column containing glucan and a column gel column containing-N ⁺ (CH ₃ ) ₃ Anion exchange column treatment with porous hydrophilic polymethacrylate as matrix to remove impurities and target eggConcentration of white, then use of a containing-N ⁺ (CH ₃ ) ₃ High performance anion chromatography of the non-porous resin filler with functional groups is used for detection and analysis. The method has simple operation and few steps, and can rapidly and accurately carry out qualitative and quantitative analysis on albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin and other proteins in urine.

Description

Analysis method for simultaneously detecting multiple proteins in urine

Technical Field

The invention relates to the field of protein extraction and purification, in particular to an analysis method for simultaneously detecting a plurality of proteins in urine.

Background

Early kidney disease screening often requires detection of markers in urine such as urinary albumin (HSA), urinary Transferrin (TRF), urinary retinol binding protein (RBP 4), urinary α1 microglobulin (A1M), urinary β2 microglobulin (B2M), NAG, which are sensitive indicators of early injury to glomeruli, tubules, proximal tubules, and early diabetic nephropathy. In clinical diagnostic procedures, various urine proteins often need to be detected in one pass for screening and diagnosis. However, the method is limited by a detection method, each marker needs to be detected independently, so that time and labor are wasted, the cost is high, sample pollution and operation errors are more easily caused due to large operation amount, and the detection result is inaccurate, so that the accurate diagnosis cannot be performed.

From the results of literature investigation, the difficulty of analyzing and measuring one to three proteins with larger differences in physicochemical properties in urine is lower, but more complex separation techniques are required for separating multiple proteins in urine at the same time. At present, the common main techniques include electrophoresis technology, highly integrated protein biochemical analysis technology, chemiluminescence technology, mass spectrometry technology and the like, wherein the electrophoresis technology has the defects of complex operation, multiple interference factors, unstable result and incapability of quantitative analysis; the turbidity of biochemical analysis technology can cause inaccuracy of quantitative results, and different kinds of proteins usually need to be detected respectively; the chemiluminescence technology is complex and the cost is high; mass spectrometry instruments and equipment are expensive and depend on importation, so that the difficulty in use and operation is high.

Thus, there is a need for an assay that can simply, rapidly, economically, and accurately detect and isolate multiple proteins in urine at the same time.

Disclosure of Invention

In order to solve the technical problems, the invention provides an analysis method capable of simultaneously detecting a plurality of proteins in urine, which is simple and convenient for treating urine samples, and can rapidly and accurately detect proteins such as albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin and the like in urine.

The invention adopts the following technical scheme:

s1, filtering: filtering urine with hydrophilic MCE filter membrane to obtain filtrate 1;

s2, first solid phase extraction: processing the filtrate 1 by a volume column gel column containing glucan to obtain filtrate 2;

s3, the firstSecondary solid phase extraction: passing the filtrate 2 to contain-N ⁺ (CH ₃ ) ₃ Treating the porous hydrophilic polymethacrylate with the functional group serving as a matrix by an anion exchange column to obtain filtrate 3;

s4, high-performance anion liquid chromatography analysis: by a method comprising-N ⁺ (CH ₃ ) ₃ Anion exchange column detection filtrate 3 of non-porous resin filler with functional group adopts an elution mode containing Cl ^- Tris gradient mobile phase of (c).

Further, the pore size of the hydrophilic MCE filter was 0.22 microns.

Further, the volume column gel column is Hitrap ^TM Desalting，5 mL，cytiva。

Further, the specific method for the first solid phase extraction is as follows:

replacement: the ethanol solution in the volume column gel column was completely replaced with 25 mL Tris-HCl buffer a (20 mM Tris-HCl, ph=8.0)

Balance: 5.0 mL Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0)

Loading: said filtrate 1 in 1.5 mL

Eluting: 2.0 mL of Tris-HCl buffer solution C, and collecting eluent to obtain filtrate 2

Flow rate: 1.0-10.0 mL/min

Column temperature: room temperature.

Further, the anion exchange column of the second solid phase extraction is one of Monomix Mab60-Q, monomix HC60-Q-II and Polar MC 60-Q9.

Further, the specific method for the second solid phase extraction is as follows:

activating: 3.0 mL of ultrapure water

Balance: 1.5 mL of the Tris-HCl buffer A

Loading: the filtrate 2

Leaching: 1.5 mL Tris-HCl buffer D (20 mM Tris-HCl,50 mM NaCl,pH =8.0)

Eluting: 0.15 mL Tris-HCl buffer B (20 mM Tris-HCl,500 mM NaCl,pH =8.0), and collecting eluent to obtain filtrate 3

Flow rate: not more than 1 d/s

Column temperature: room temperature.

Further, the nonporous resin is polystyrene/divinylbenzene (PS/DVB) resin particles with the particle diameter of 1.7-10 mu m, and the surface is bonded with a hydrophilic polymer nano thin layer, and the nano thin layer is modified with the-N ⁺ (CH ₃ ) ₃ Functional groups.

Further, the high performance anion liquid chromatography column is Proteomix SAX NP5, 4.6X10 mm, sepax.

Further, the specific method for high performance anion liquid chromatography analysis comprises the following steps:

mobile phase: said buffer A and said buffer B

Loading: the filtrate 3

Elution gradient:

0-0.5min 0%B

0.5-7.0min 0-100%B

7.0-7.1min 100-0%B

7.1-15min 0%B

a detector: ultraviolet detection wavelength 210, 280nm

Column temperature: room temperature

Flow rate: 0.5 mL/min.

Further, the assay primarily detects one or more of albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin in urine.

The urine sample needs to be subjected to pretreatment by filtration and solid phase extraction, because the urine contains more impurities and 5 target proteins with low content, and most of impurities (mainly small molecules) are removed and target proteins are enriched by pretreatment. Firstly, filtering urine by using a needle filter (a hydrophilic filter membrane with the pore diameter of 0.22 microns) to remove sediment, bacteria and the like; then, by utilizing the volume difference of target protein and small molecular impurities, selecting a dextran gel column based on a volume exclusion principle to remove the small molecular impurities, and simultaneously replacing urine to a low salt concentration and a weak alkaline environment so as to prepare for subsequent concentration; and then, utilizing isoelectric point difference of the target protein and other proteins, selecting an anion exchange column, selectively adsorbing the target protein, eluting the target protein by a small volume of liquid, and completing concentration enrichment and further impurity removal.

And (3) carrying out high-efficiency anion liquid chromatography analysis on the urine sample subjected to filtration and twice solid phase extraction, wherein a stationary phase filler is positively charged under the environment of pH=8.0, albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin are negatively charged, and binding force such as electrostatic interaction exists between the stationary phase and the protein. Because the isoelectric points of the five proteins are different, the electrostatic acting force between the fixed phase and the five proteins is also different. In the gradient elution process, the proportion of the mobile phase B is continuously increased, and Cl in the mobile phase B ^- The five proteins compete with the five proteins to bind to the stationary phase, so that the five proteins can flow out sequentially from weak binding force to strong binding force, and further qualitative and quantitative detection is completed.

Compared with the prior art, the analysis method for simultaneously detecting multiple proteins in urine provided by the invention is simple in operation and few in steps, and can rapidly and accurately perform qualitative and quantitative analysis on proteins such as albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin and the like in urine.

Drawings

FIG. 1 is a graph of the detection wavelength at 210 nm of a raw urine sample 1 after filtration through a 0.22 micron membrane;

FIG. 2 is a graph of the detection wavelength at 280nm of a raw urine sample 1 after filtration through a 0.22 micron membrane;

FIG. 3 is a graph of the detection wavelength at 210 nm of a raw urine sample 1 after 0.22 micron membrane filtration and a first solid phase extraction;

FIG. 4 is a graph of the detection wavelength at 280nm of a raw urine sample 1 after 0.22 micron membrane filtration and a first solid phase extraction;

FIG. 5 is a graph of the detection wavelength at 210 and 280nm of the original urine sample 1 after filtration, two solid phase extractions and high performance anion chromatography;

FIG. 6 is an enlarged spectrum of detection wavelengths at 210 and 280nm of the original urine sample 1 after filtration, two solid phase extractions, and high performance anion chromatography;

FIG. 7 is a graph of the detection wavelength at 210 and 280nm of the raw urine sample 2 after filtration, two solid phase extractions, and high performance anion chromatography;

FIG. 8 is an enlarged spectrum of detection wavelengths at 210 nm of the original urine sample 2 after filtration, two solid phase extractions, and high performance anion chromatography;

FIG. 9 is an enlarged spectrum of detection wavelength at 280nm of the original urine sample 2 after filtration, twice solid phase extraction, high performance anion chromatography;

FIG. 10 is a graph of the detection wavelength spectra of beta 2 microglobulin (B2M) single standard samples at 210 and 280 nm;

FIG. 11 is a graph of the detection wavelength spectrum of a single sample of Transferrin (TRF) at 210 and 280 nm;

FIG. 12 is a graph of the detection wavelength spectra of retinol binding protein (RBP 4) single standard samples at 210 and 280 nm;

FIG. 13 is a graph of the detection wavelength spectra of α1 microglobulin (A1M) single standard samples at 210 and 280 nm;

fig. 14 is a graph of detection wavelength spectra of albumin (HSA) single standard samples at 210 and 280 nm.

Detailed Description

The following description of embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is shown, however, only some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Eluent preparation

Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0) was prepared: 2.4228 g Tris (hydroxymethyl) aminomethane (Tris) was weighed, dissolved in 1L ultrapure water, adjusted to pH 8.0 using hydrochloric acid (HCl), and filtered through a 0.45 μm hydrophilic filter membrane.

Tris-HCl buffer B (20 mM Tris-HCl,500 mM NaCl,pH =8.0) was prepared: 2.4228 g Tris (hydroxymethyl) aminomethane (Tris) and 29.2214 g sodium chloride (NaCl) were weighed, dissolved in 1L ultrapure water, adjusted to pH 8.0 with hydrochloric acid (HCl), and filtered through a 0.45 μm hydrophilic filter membrane.

Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0) was prepared: tris-HCl buffer A and Tris-HCl buffer B were mixed in a 95:5 ratio.

Tris-HCl buffer D (20 mM Tris-HCl,50 mM NaCl,pH =8.0) was prepared: tris-HCl buffer A and Tris-HCl buffer B were mixed in a ratio of 90:10.

Example 1

1) Filtration

The raw urine sample 1 was filtered using a needle filter (hydrophilic filter membrane with 0.22 μm pore size), and the filtrate 2 of the raw urine was collected.

Figures 1 and 2 show graphs of filtrate 2 after needle filtration at 210 nm and 280nm, and it can be seen that the urine component after 0.22 micron membrane filtration is still very complex and requires further removal of impurities.

2) First solid phase extraction

Column selection: hitrap ^TM Desantng, 5 mL, cytova (Sephadex G-25 Superfine packed Sephadex column)

Replacement: for the first time, it was necessary to replace the ethanol solution in the sephadex column completely with 25 mL of Tris-HCl buffer a (20 mM Tris-HCl, ph=8.0)

Balance: 5.0 mL Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0)

Loading: 1.5 mL urine filtrate 2

And (3) collecting: after loading was completed, 2.0 mL of Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0) was added and effluent 3 was collected

Cleaning: 10.0 mL Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0)

Flow rate: 1.0-10.0 mL/min

Column temperature: room temperature.

Figures 3 and 4 show spectra of urine 1 (i.e., effluent 3) at 210 nm and 280nm after a first solid phase extraction, it can be seen that most small molecular substances are removed and interfering components are significantly reduced after the first solid phase extraction.

3) Second solid phase extraction

Column selection: an anion exchange column packed with Monomix Mab60-Q,50mg, sepax filler, or packed with Monomix HC60-Q-II, polar MC60-Q9, etc. may be used. These fillers are based on porous hydrophilic polymethacrylates and contain-N on the matrix ⁺ (CH ₃ ) ₃ Functional groups.

Activating: 3.0 mL of ultrapure water

Balance: 1.5 mL Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0)

Loading: effluent 3 after the first solid phase extraction

Leaching: 1.5 mL Tris-HCl buffer D (20 mM Tris-HCl,50 mM NaCl,pH =8.0)

Eluting: 0.15 The sample 4 is obtained by collecting eluent which is the mL Tris-HCl buffer B (20 mM Tris-HCl,500 mM NaCl,pH =8.0)

Cleaning: 3.0 mL Tris-HCl buffer A (20 mM Tris-HCl, pH=8.0)

Flow rate: not more than 1 d/s

Column temperature: room temperature.

And performing secondary solid phase extraction to complete ten times concentration of the target protein.

4) High performance liquid chromatography

Mobile phase E was configured:

2.4228 g Tris (hydroxymethyl) aminomethane (Tris) was weighed, dissolved in 1L ultrapure water, pH was adjusted to 8.0 using hydrochloric acid (HCl), and filtered through a 0.45 μm hydrophilic filter membrane to give mobile phase E.

Mobile phase F:

2.4228 g Tris (hydroxymethyl) aminomethane (Tris) and 29.2214 g sodium chloride (NaCl) were weighed, dissolved in 1L of ultra pure water, adjusted to pH 8.0 with hydrochloric acid (HCl), and filtered through a 0.45 μm hydrophilic filter to give mobile phase F.

Column selection: proteomix SAX NP5, 4.6X10 mm, sepax, anion exchange analytical column

Mobile phase E:20 mM Tris-HCl, pH=8.0

Mobile phase F:20 mM Tris-HCl,500 mM NaCl,pH =8.0

Elution gradient:

0-0.5min 0%F

0.5-7.0min 0-100%F

7.0-7.1min 100-0%F

7.1-15min 0%F

a detector: ultraviolet detection wavelength 210, 280nm

Column temperature: room temperature

Flow rate: 0.5 mL/min

Sample injection volume: 20. mu.L of sample 4 was injected.

The filtered and twice solid-phase extracted raw urine sample 1 is analyzed by adopting the high-performance anion liquid chromatography method in the step 4), and the analysis result is shown in fig. 5-6. Qualitative analysis was completed by comparison with the peak time of each single-labeled protein in comparative example 1, and quantitative analysis was completed by comparison with the peak area size of each protein, and the analysis results are shown in table 1.

TABLE 1

The comparison analysis shows that the peak area of the beta 2 microglobulin B2M in the original urine sample 1 is larger than the upper limit value 12.2 of the beta 2 microglobulin B2M in the urine of normal people, which indicates that the B2M content exceeds the upper limit of the normal reference, the other 4 protein contents are smaller than the upper limit of the normal reference interval, and the increase of the beta 2 microglobulin B2M in the urine generally indicates the damage of the renal tubular function or the increase of the filtration load.

Example 2

Unlike example 1, which uses a raw urine sample 2, example 2 uses the same raw urine sample as example 1, and the analysis results are shown in FIGS. 7 to 9.

Qualitative analysis was completed by comparison with the peak time of each single-labeled protein in comparative example 1, and quantitative analysis was completed by comparison with the peak area size of each protein, and the analysis results are shown in table 2.

TABLE 2

In the original urine sample 2, the peak area of the B2M peak is 51.7 (280 nm), the peak area of the TRF peak is 56.0 (280 nm), the peak area of the RBP4 peak is 31.4 (280 nm), the peak area of the A1M peak is 94.1 (280 nm), the peak area of the HSA peak is 322.1 (280 nm), and the comparison analysis shows that the 5 protein contents exceed the upper limit of the normal reference interval, which indicates that the renal function of the patient can be obviously severely damaged.

In the step 4), the filler adopted by the anion exchange analysis column Proteomix SAX NP5 is rigid, spherical and non-porous polystyrene/divinylbenzene (PS/DVB) resin particles with high crosslinking degree, the particle size can be 1.7 mu m, 3 mu m, 5 mu m and 10 mu m, preferably 5 mu m, the surface of the resin is bonded with a neutral polymer thin layer with high hydrophilic nanoscale thickness, and the surface of the hydrophobic PS/DVB resin is completely covered by the hydrophilic material, so that irreversible adsorption of PS/DVB to biomolecules is eliminated, and the separation efficiency and the biological sample recovery rate are high; the surface of the polymer thin layer is densely and uniformly chemically bonded with a strong anion exchange functional group (quaternary amino group). The stationary phase filler has three characteristics: firstly, the nanoscale thickness of the hydrophilic layer completely eliminates non-specific interactions between the carrier and the biological sample; secondly, the non-porous particle structure minimizes lateral diffusion of the sample while inhibiting its diffusion into the filler particles; thirdly, by using the unique chemical bonding technology of the siren, three-dimensional strong anion exchange groups are bonded on the hydrophilic layer, and the best resolution and separation efficiency can be provided for proteins, oligonucleotides, saccharides, polypeptides and the like.

Since the stationary phase filler is positively charged, and the isoelectric points (Isoelectric Point, pI) of albumin, transferrin, retinol binding protein, beta 2 microglobulin and alpha 1 microglobulin in urine are all less than 8, the proteins are in the environment of pH=8.0The released protons are negatively charged, so that the stationary phase and the protein have binding force such as electrostatic action. Because the isoelectric points of the five proteins are different, the magnitudes of electrostatic acting forces between the fixed phase and the five proteins are also different. In the gradient elution process, the proportion of the mobile phase F is continuously increased, and Cl in the mobile phase F ^- Ions compete with the five proteins to bind to the stationary phase, so that the five proteins can flow out sequentially from weak binding force to strong binding force, and separation is completed. Typically, the order of protein efflux is from high to low isoelectric point.

TABLE 3 Table 3

Comparative example 1

Preparing each protein sample:

mu.L of 1.0 mg/mL of beta 2 microglobulin (B2M), 1.4 mu.L of 1.0 mg/mL of retinol binding protein (RBP 4), 2.0 mu.L of 2.0 mg/mL of Transferrin (TRF), 20 mu.L of 2.0 mg/mL of albumin (HSA), 12 mu.L of 2.0 mg/mL of alpha 1 microglobulin (A1M) were each pipetted using a pipette and the mobile phase E was used as a diluent to a volume of 200 mu.L, respectively, to prepare five protein single-label samples.

The concentrations of the five protein single standard samples are respectively as follows: the concentration of the beta 2 microglobulin (B2M) sample is 3.0 mug/mL, which corresponds to the upper limit of the urine reference interval of normal people of 0.3 mug/mL; transferrin (TRF) sample concentration is 20.0 μg/mL, corresponding to the upper limit of 2.0 μg/mL of the urine reference interval of normal people; retinol binding protein (RBP 4) sample concentration is 7.0 mug/mL, corresponding to the upper limit of 0.7 mug/mL of the urine reference zone of normal human; albumin (HSA) sample concentration is 200.0 μg/mL, corresponding to the upper limit of 20.0 μg/mL of the normal human urine reference interval; the sample concentration of the alpha 1 microglobulin (A1M) is 120.0 mug/mL, which corresponds to the upper limit of 12.0 mug/mL of the urine reference zone of a normal person.

The five protein single-label samples are analyzed by the high-performance anion liquid chromatography method in the step 4), and the analysis results are shown in figures 10-14. The peak time of beta 2 microglobulin (B2M) is as follows: 3.736 min, peak area 201 (210 nm), 12.2 (280 nm); the peak time of Transferrin (TRF) is: 4.182 min, peak area 871.4 (210 nm), 46.7 (280 nm); retinol binding protein (RBP 4) peak time 4.681 min, peak area 314.9 (210 nm), 20.9 (280 nm); the peak time of the alpha 1 microglobulin (A1M) is as follows: 4.932 min, peak area is: 1035 (210 nm), 78.4 (280 nm); the peak time of albumin (HSA) was: 5.493 min, peak area is: 11068.5 (210 nm), 292.8 (280 nm).

The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.

Claims

1. An analytical method for simultaneously detecting a plurality of proteins in urine, comprising the steps of:

1) And (3) filtering: filtering urine with hydrophilic MCE filter membrane to obtain filtrate 1;

2) First solid phase extraction: processing the filtrate 1 by a volume column gel column containing glucan to obtain filtrate 2;

3) Second solid phase extraction: passing the filtrate 2 to contain-N ⁺ (CH ₃ ) ₃ Treating the porous hydrophilic polymethacrylate with the functional group serving as a matrix by an anion exchange column to obtain filtrate 3;

4) High performance anion liquid chromatography analysis: by a method comprising-N ⁺ (CH ₃ ) ₃ Anion exchange column detection filtrate 3 of non-porous resin filler with functional group adopts an elution mode containing Cl ^- Tris gradient mobile phase of (c).

2. The method of claim 1, wherein the protein is one or more of albumin, transferrin, retinol binding protein, beta 2 microglobulin, alpha 1 microglobulin.

3. The method of claim 1, wherein the hydrophilic MCE filter has a pore size of 0.22 microns.

4. The method for simultaneously detecting multiple proteins in urine according to claim 1, wherein the volume column gel column is Hitrap ^TM Desalting，5 mL，cytiva。

5. The method for simultaneously detecting multiple proteins in urine according to claim 4, wherein the specific method for the first solid phase extraction is as follows:

Balance: 5.0 mL Tris-HCl buffer C (20 mM Tris-HCl,25 mM NaCl,pH =8.0)

Loading: said filtrate 1 in 1.5 mL

Flow rate: 1.0-10.0 mL/min

Column temperature: room temperature.

6. The method according to claim 1, wherein the anion exchange column of the second solid phase extraction is one of Monomix Mab60-Q, monomix HC60-Q-II and Polar MC 60-Q9.

7. The method for simultaneously detecting multiple proteins in urine according to claim 6, wherein the specific method for the second solid phase extraction is as follows:

activating: 3.0 mL of ultrapure water

Balance: 1.5 mL of the Tris-HCl buffer A

Loading: the filtrate 2

Leaching: 1.5 mL Tris-HCl buffer D (20 mM Tris-HCl,50 mM NaCl,pH =8.0)

Flow rate: not more than 1 d/s

Column temperature: room temperature.

8. The method according to claim 1, wherein the nonporous resin is polystyrene/divinylbenzene (PS/DVB) resin particles having a particle size of 1.7 to 10 μm, and is surface-bonded with a hydrophilic polymer nano-sheet, the nano-sheet being modified with the-N ⁺ (CH ₃ ) ₃ Functional groups.

9. The method according to claim 8, wherein the high performance anion liquid chromatography column is Proteomix SAX NP5, 4.6X50 mm, sepax.

10. The method for simultaneously detecting multiple proteins in urine according to claim 9, wherein the specific method for high performance anionic liquid chromatography is as follows:

mobile phase: said buffer A and said buffer B

Loading: the filtrate 3

Elution gradient:

0-0.5min 0%B

0.5-7.0min 0-100%B

7.0-7.1min 100-0%B

7.1-15min 0%B

a detector: ultraviolet detection wavelength 210, 280nm

Column temperature: room temperature

Flow rate: 0.5 mL/min.